U.S. patent application number 10/726321 was filed with the patent office on 2005-06-02 for analyte identification in transformed electropherograms.
Invention is credited to Burgi, Dean, Williams, Stephen.
Application Number | 20050115837 10/726321 |
Document ID | / |
Family ID | 34620502 |
Filed Date | 2005-06-02 |
United States Patent
Application |
20050115837 |
Kind Code |
A1 |
Burgi, Dean ; et
al. |
June 2, 2005 |
Analyte identification in transformed electropherograms
Abstract
The present invention is directed to methods for identifying one
or more analytes in a sample using electrophoresis. In one
embodiment, the method comprises performing an electrophoretic
separation by applying a potential across the separation path and
thus generating a current and power therein and producing an
electropherogram, integrating the current or the power to determine
the cumulative current or power as a function of the separation
time, transforming the electropherogram to a second
electropherogram representing the signal as a function of the
cumulative current or power, and identifying in the second
electropherogram peaks that are correlated with the analytes in the
sample. The invention also provides systems for performing the
analysis and identification methods, as well as computer-readable
products for performing the steps associated with the above
methods.
Inventors: |
Burgi, Dean; (Sunnyvale,
CA) ; Williams, Stephen; (San Carlos, CA) |
Correspondence
Address: |
ACLARA BIOSCIENCES, INC.
1288 PEAR AVENUE
MOUNTAIN VIEW
CA
94043
US
|
Family ID: |
34620502 |
Appl. No.: |
10/726321 |
Filed: |
December 1, 2003 |
Current U.S.
Class: |
204/452 ;
204/603 |
Current CPC
Class: |
G01N 27/44704 20130101;
G01N 27/44773 20130101; G01N 27/4473 20130101; G01N 27/44717
20130101 |
Class at
Publication: |
204/452 ;
204/603 |
International
Class: |
G01N 027/453 |
Claims
What is claimed is:
1. A method of identifying one or more analytes in a sample by
electrophoretic separation, the method comprising the steps of:
applying a potential across a separation path containing one or
more analytes to generate a current therein and to separate the one
or more analytes so that a first electropherogram of a signal as a
function of time is produced; integrating the current with respect
to time to provide a cumulative current as a function of time;
transforming the first electropherogram to a second
electropherogram of the signal as function of the cumulative
current; and identifying in the second electropherogram peaks that
are correlated with the one or more analytes in the sample.
2. The method according to claim 1, wherein said potential is
constant.
3. The method according to claim 1, wherein said potential varies
with time, and wherein said second electropherogram is a function
further comprised of said potential as a function of time.
4. The method according to claim 3, wherein said potential varies
with time such that said current in said separation path is
constant.
5. The method according to claim 3, wherein said potential varies
with time such that the power in said separation path is
constant.
6. The method according to claim 1, wherein said separation path is
a capillary tube.
7. The method according to claim 1, further comprising at least one
electrophoretic mobility standard in said sample, wherein the at
least one electrophoretic standard is used to identify peaks that
are correlated with said one or more analytes of said sample.
8. The method according to claim 7, comprising two electrophoretic
standards wherein the mobility of the first electrophoretic
standard is greater than that of any analyte and the mobility of
the second electrophoretic standard is less than that of any
analyte in said sample.
9. The method according to claim 8, wherein said one or more
analytes are molecular tags, wherein each tag has a different
electrophoretic mobility.
10. The method according to claim 9, wherein the presence in said
sample of said molecular tags is the result of a specific
recognition event with at least one type of biomolecule selected
from the group of proteins, antigens, receptors, DNA and RNA.
11. The method according to claim 9 or claim 10, wherein said one
or more analytes of said sample is a plurality of said molecular
tags, numbering in the range of from 2 to 50.
12. A system for identifying one or more analytes in a sample using
electrophoretic separation, the system comprising: a separation
path comprising a separation medium; a voltage source for applying
a potential across the separation path so that a current is
generated in the separation path and one or more analytes are
separated along the separation path; a detector positioned along
the separation path for recording a first electropherogram of the
signal intensity associated with the one or more analytes in the
separation path as a function of time; and a processor comprising
software for (a) integrating with respect to time the current in
the separation path to provide the cumulative current as a function
of time; (b) transforming the first electropherogram to a second
electropherogram of the signal intensity associated with the
analytes as a function of the cumulative current; and (c)
identifying in the second electropherogram peaks that are
correlated with the one or more analytes in the sample.
13. The system according to claim 12, wherein said voltage source
applies a constant voltage.
14. The system according to claim 12, wherein said voltage source
applies a voltage varying with time, and further comprising a
voltage recording device for recording the voltage applied across
said separation path as a function of time, and said processor
further comprising software for transforming said first
electropherogram to a second electropherogram of the signal
intensity as a function further comprised of the applied potential
as a function of time.
15. The system according to claim 14, wherein said voltage source
applies a voltage varying in time such that said current in said
separation path is constant.
16. The system according to claim 14, wherein said voltage source
applies a voltage varying in time such that the power in said
separation path is constant.
17. The system according to claim 12, wherein said separation path
is a capillary tube.
18. The system according to any one of claims 12 to 17, further
comprising a plurality of separation paths.
19. The system according to claim 18, wherein said voltage source
applies a potential independently across each of said separation
paths.
20. The system according to claim 18, wherein said voltage source
applies a potential jointly across said separation paths.
21. The system according to claim 12, further comprising at least
one electrophoretic mobility standard, wherein the at least one
mobility standard is used to identify in the second
electropherogram peaks that are correlated with the analytes.
22. The system according to claim 21, comprising two mobility
standards wherein the mobility of the first mobility standard is
greater than that of any analyte and the mobility of the second
mobility standard is less than that of any analyte in said
sample.
23. A computer-readable product embodying a program for execution
by a computer to identify one or more analytes in an
electrophoretic separation by determining peak locations in a
transformed electropherogram and correlating such peak locations
with the one or more analytes, the program comprising instructions
for: reading a first electropherogram data set of an analyte signal
as a function of separation time from a data storage medium;
reading a data set of current as a function of separation time from
a data storage medium; determining a data set of cumulative current
as a function of separation time; transforming the first
electropherogram data set to a second electropherogram data set of
the analyte signal as a function of cumulative current; identifying
peak locations in the second electropherogram; and correlating the
identified peak locations with each of the one or more analytes.
Description
FIELD OF THE INVENTION
[0001] This invention relates to a method and a system for
detecting and/or measuring one or more analytes in a sample by
electrophoretic separation, and more particularly, to methods for
analyzing data generated by an electrophoretic separation.
BACKGROUND OF THE INVENTION
[0002] Separation by electrophoresis is a widely used analytical
and preparative technique, especially in the life sciences.
Electrophoretic separation is based on the movement of charged
analytes in solution under the influence of an electric field. The
rate of migration of an analyte depends on the size and shape of
the analyte, the charge carried, the applied voltage and the
resistance of the separation medium, Rickwood and Hames, Gel
Electrophoresis of Nucleic Acids: A Practical Approach (IRL Press,
Oxford, 1982). Many variations of the technique have been developed
depending on the class of analyte being examined, e.g. DNA,
proteins, small molecule drugs, and the like. In particular,
capillary electrophoresis has developed into an important
analytical technique that finds wide applications in DNA sequencing
technologies, quality control systems, forensics, and the like, for
measuring many different kinds of analytes, including,
polynucleotides, proteins, and small organic molecules.
[0003] The popularity of capillary electrophoresis is based on
several important technical advantages: (i) capillaries have high
surface-to-volume ratios which permit more efficient heat
dissipation which, in turn, permit the application of high electric
fields for more rapid separations; (ii) the technique requires
minimal sample volumes; (iii) high resolution of most analytes is
attainable; and (iv) the technique is amenable to automation, e.g.
Camilleri, editor, Capillary Electrophoresis: Theory and Practice
(CRC Press, Boca Raton, 1993); Grossman et al, editors, Capillary
Electrophoresis (Academic Press, San Diego, 1992); and Landers,
editor, Handbook of Capillary Electrophoresis, Second Edition (CRC
Press, Boca Raton, 1997).
[0004] The results of electrophoretic analysis are frequently
provided as an electropherogram that depicts a record of signal
intensity values versus time, or versus position in some cases.
That is, an electropherogram is a graphical representation of
signal intensity as a function of time or position. The data in an
electropherogram may be collected in a variety of ways, depending
on the type of electrophoretic technique employed and the type of
signal detected. In many electrophoretic systems, a signal is
collected at a particular station along the separation path, as
shown in FIG. 1A, which is a diagram illustrating the main
components of a capillary electrophoresis system. In a successful
separation, sample constituents form distinct peaks of various
heights and widths in an electropherogram.
[0005] A problem often encountered with electrophoresis is that the
same sample constituents may appear on an electropherogram at
different migration times for different samples of the same kind.
That is, a constituent, or analyte, common to two different samples
may appear at a different place on each of the electropherograms
for such samples. Factors that contribute to such variability
include changes in the migration rates of the constituents caused
by changes in the local environments of the analytes during a
separation, perhaps caused by the introduction of the sample
itself. That is, the process of separating multiple constituents of
a sample from one another can affect the local conductivity of the
separation medium around the constituents; and hence, their
migration rates.
[0006] This creates a difficulty in many analytical procedures
since analytes are typically identified either (i) by the
appearance of a peak of a particular size or position on an
electropherogram relative to the peaks of other sample constituents
or relative to the peak(s) of a standard or (ii) by a
characteristic migration time under predetermined separation
conditions. In either case, local variations in analyte migration
rates reduce the accuracy of such identification. These
difficulties can be particularly troublesome in the separation of
complex samples, where large numbers of analytes are sought to be
identified in a single separation path, such as fragment ladders in
DNA sequencing, and multiplexed analytical techniques, e.g. Singh
et al, International patent publications WO 00/66607; WO 01/83502;
WO 02/95356; WO 03/06947; and U.S. Pat. Nos. 6,322,980 and
6,514,700.
[0007] Performing electrophoretic separations at constant power
provides one means of improving temperature uniformity and reducing
fluctuations and variations in the migration rates. However, the
majority of commercial electrophoresis instruments, particularly
those for capillary electrophoresis operate in constant voltage
mode. In these cases the end-user has no recourse for improving the
analytical performance via the hardware.
[0008] In view of the above, the availability of a convenient
method for accounting for and correcting the affects of varying
analyte migration rates would advance many fields where
electrophoretic separations are important, including life science
research, medical research and diagnostics, forensics, and the
like.
SUMMARY OF THE INVENTION
[0009] The present invention is directed to a method, system and
product for identifying one or more analytes in a sample using
electrophoresis. In one aspect, the method comprises the steps of
(a) applying a potential across a separation path containing one or
more analytes to generate a current therein and to produce an
electropherogram of the one or more analytes, (b) integrating the
current to determine the cumulative current as a function of the
separation time, (c) transforming the electropherogram to a second
electropherogram representing the signal as a function of the
cumulative current, and (d) identifying in the second
electropherogram peaks that are correlated with the analytes in the
sample.
[0010] In another aspect, the method comprises the steps of
performing an electrophoretic separation by applying a potential
across the separation path containing one or more analytes thereby
generating an electrical power therein and producing an
electropherogram, integrating the power to determine the cumulative
power as a function of the separation time, transforming the
electropherogram to a second electropherogram representing the
signal as a function of the cumulative power, and identifying in
the second electropherogram peaks that are correlated with the
analytes in the sample.
[0011] In yet another aspect, a plurality of separation paths is
provided for the identification of one or more analytes in a
plurality of samples. In one embodiment, a potential is applied
independently across each of the plurality of separation paths, and
in another embodiment a potential is applied jointly across the
entire plurality of separation paths. In either embodiment,
electropherograms are produced for each separation path, the
current is each path is integrated to provide the cumulative
current as a function of time for each path, each electropherogram
is transformed to a respective second electropherogram representing
the signal as a function of the cumulative current, and finally
peaks in the second electropherograms are identified by correlation
with the analytes in the samples.
[0012] In another aspect, the invention provides a method for
identifying analytes in a sample separated by electrophoresis to
give a first data set of a signal as a function of time and a data
set of the separation path power or current as a function of time,
wherein the method comprises the steps of integrating the
separation path parameter (power or current) with respect to time
to provide a cumulative parameter as a function of time,
transforming the first signal data to a second data set of the
signal as a function of the cumulative parameter (power or
current), and identifying in the second data set peaks correlated
with the analytes in the sample.
[0013] In another aspect, the invention provides a system for
performing the above methods. In one embodiment, the system
comprises a separation path comprising a separation medium, a
voltage source for applying a potential across the length of the
separation path wherein a current and power are generated, a
detector positioned along the separation path for recording a first
electropherogram of the signal intensity associated with the
analytes as a function of the separation time, and a processor
comprising software for (a) integrating with respect to the
separation time the current in the separation path to provide the
cumulative current as a function of time; (b) transforming the
first electropherogram to a second electropherogram of the signal
intensity associated with the analytes as a function of the
cumulative current; and (c) identifying in the second
electropherogram peaks that are correlated with the analytes in the
separation path.
[0014] In any of the aforementioned embodiments the separation path
may comprise a capillary tube, capillary channel, microfluidic
channel, or the like, as typically found in systems known and
disclosed in the art. Automated capillary array electrophoresis
instruments are a convenient means for performing the
electrophoretic separation. Also, in another aspect of the
aforementioned embodiments, the samples further comprise at least
one electrophoretic mobility standard.
[0015] In another aspect, the invention provides computer-readable
products for performing steps of the above methods.
[0016] In any embodiment of the invention, the one or more analytes
may be molecular tags, wherein each tag has a different
electrophoretic mobility, wherein the presence of the molecular
tags in the sample is the result of a specific recognition event
with at least one type of molecule selected from the group of
proteins, antigens, receptors, DNA and RNA, and wherein the number
of types of such molecular tags range from 2 to 50.
[0017] The present invention provides a method, system and product
for identifying, detecting or measuring one or more analytes that
has several advantages over current techniques including, but not
limited to, (1) accurate detection and quantification of peaks in
electropherogram data, and (2) consistent electrophoretic analyses
to overcome run-to-run, channel-to-channel and
instrument-to-instrument variation, by correcting for fluctuations
in the separation conditions that would otherwise cause
fluctuations in the observed migration times or distances of
analytes. The invention may also be employed to up-grade existing
instruments for electrophoresis to give them the favorable
properties of a constant power separation instrument without the
need for expensive hardward alterations.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1A is a diagram illustrating the main components of an
instrument for conducting capillary electrophoresis.
[0019] FIG. 1B is a diagram illustrating the main components of a
system for conducting slab gel electrophoresis.
[0020] FIGS. 1C through 1E illustrate steps in practicing an
electrophoretic separation using a microfluidics capillary
electrophoresis (CE) device.
[0021] FIG. 2A is a flow chart illustrating the steps of an
embodiment of the invention for identifying analytes in an
electrophoretic separation.
[0022] FIGS. 2B and 2C are illustrations of embodiments of a system
for performing the invention.
[0023] FIG. 2D illustrates the component functions of a
computer-readable product for performing the invention.
[0024] FIGS. 3A through 3K illustrate features of a peak
identification algorithm for use with the invention.
[0025] FIG. 4 is a flow chart illustrating the steps of an
algorithm for identifying peaks in electropherogram data.
[0026] FIG. 5A illustrates an exemplary multiplexed assay for
detecting or measuring target analytes, such as proteins, by
generating molecular tags in a "sandwich" type of assay using
antibodies as binding compounds.
[0027] FIG. 5B illustrates an exemplary multiplexed assay for
detecting or measuring target polynucleotides by generating
molecular tags in a "taqman" type of assay in a polymerase chain
reaction (PCR).
[0028] FIG. 5C illustrates an exemplary multiplexed assay for
detecting or measuring target polynucleotides by generating
molecular tags in an Invader type of assay.
[0029] FIGS. 6A and 6B illustrate the chemical formulas of ten
molecular tags.
[0030] FIG. 7A shows a set of electropherograms of signal versus
time.
[0031] FIG. 7B shows the data of FIG. 7A as a set of
electropherograms of signal versus relative migration time with
respect to electrophoretic standards.
[0032] FIG. 7C shows the data of FIG. 7A as a set of
electropherograms transformed according to one embodiment of the
invention.
[0033] FIG. 8 is an electropherogram showing peaks identified
according to molecular tag and associated analyte.
DEFINITIONS
[0034] "Analyte" in the present specification and claims is used in
a broad sense. On the one hand, the term means a substance,
compound, or component in a sample whose presence or absence is to
be detected or whose quantity is to be measured in an assay. In
such a case, "target" may be used interchangeably with "analyte".
Analytes include but are not limited to peptides, proteins,
polynucleotides, polypeptides, oligonucleotides, organic molecules,
haptens, epitopes, parts of biological cells, posttranslational
modifications of proteins, receptors, complex sugars, vitamins,
hormones, and the like. There may be more than one analyte
associated with a single molecular entity, e.g. different
phosphorylation sites on the same protein, different SNP's within a
gene, etc. On the other hand, "analyte" is also used to mean the
components of a sample that are subjected to electrophoretic
separation analysis. The one or more components, or "analytes" of a
sample are separated and detected by the analysis. In one aspect of
the present invention, the common terms are linked in the following
manner: an assay is performed on a biological "sample" to test for
the presence or amount of one or more "analytes" (targets) by
employing analyte-specific probes labeled with molecular tags. In
the assay reaction, the binding of probe to analyte is followed by
the release of the molecular tags. The result of the assay is
determined by electrophoresis using a "sample" of the assay
solution to determine the presence or amount of the molecular tag
"analytes" as found in the electropherogram. Because the
composition of the analyte-specific probes labeled with molecular
tags are known, the presence of a certain molecular tag "analyte"
in an electropherogram directly correlates with the presence of the
targeted biological "analyte" in the sample.
[0035] "Antibody" means an immunoglobulin that specifically binds
to, and is thereby defined as complementary with, a particular
spatial and polar organization of another molecule. The antibody
can be monoclonal or polyclonal and can be prepared by techniques
that are well known in the art such as immunization of a host and
collection of sera (polyclonal) or by preparing continuous hybrid
cell lines and collecting the secreted protein (monoclonal), or by
cloning and expressing nucleotide sequences or mutagenized versions
thereof coding at least for the amino acid sequences required for
specific binding of natural antibodies. Antibodies may include a
complete immunoglobulin or fragment thereof, which immunoglobulins
include the various classes and isotypes, such as IgA, IgD, IgE,
IgG1, IgG2a, IgG2b and IgG3, IgM, etc. Fragments thereof may
include Fab, Fv and F(ab').sub.2, Fab', and the like. In addition,
aggregates, polymers, and conjugates of immunoglobulins or their
fragments can be used where appropriate so long as binding affinity
for a particular polypeptide is maintained.
[0036] "Antibody binding composition" means a molecule or a complex
of molecules that comprise one or more antibodies and derives its
binding specificity from an antibody. Antibody binding compositions
include, but are not limited to, antibody pairs in which a first
antibody binds specifically to a target molecule and a second
antibody binds specifically to a constant region of the first
antibody; a biotinylated antibody that binds specifically to a
target molecule and streptavidin derivatized with moieties such as
molecular tags or photosensitizers; antibodies specific for a
target molecule and conjugated to a polymer, such as dextran,
which, in turn, is derivatized with moieties such as molecular tags
or photosensitizers; antibodies specific for a target molecule and
conjugated to a bead, or microbead, or other solid phase support,
which, in turn, is derivatized with moieties such as molecular tags
or photosensitizers, or polymers containing the latter.
[0037] "Binding compound" means any molecule to which molecular
tags can be directly or indirectly attached that is capable of
specifically binding to a membrane-associated analyte. Binding
compounds include, but are not limited to, antibodies, antibody
binding compositions, peptides, proteins, particularly secreted
proteins and orphan secreted proteins, nucleic acids, and organic
molecules having a molecular weight of up to 1000 daltons and
consisting of atoms selected from the group consisting of hydrogen,
carbon, oxygen, nitrogen, sulfur, and phosphorus.
[0038] "Capillary" refers to a tube or channel or other structure
capable of supporting a volume of separation medium for carrying
out electrophoresis. The geometry of a capillary may vary widely
and includes tubes with circular, semi-circular, rectangular or
square cross-sections, channels, grooves, plates and the like, and
may be fabricated by a wide range of technologies. An important
feature of a capillary for use with the invention is the
surface-to-volume ratio of the surface in contact with the volume
of separation medium. High values of this ratio permit better heat
transfer from the separation medium during electrophoresis.
Preferably, capillaries for use with the invention are made of
silica, fused silica, quartz, silicate-based glass, such as
borosilicate glass, phosphate glass, and the like, or other
silica-like materials.
[0039] "Capillary-sized" in reference to a separation column means
a capillary tube or channel in a plate or microfluidics device,
where the diameter or largest dimension of the separation column is
between about 25-500 microns, allowing efficient heat dissipation
throughout the separation medium, with consequently low thermal
convection within the medium.
[0040] "Computer-readable product" means any tangible medium for
storing information that can be read by or transmitted into a
computer. Computer-readable products include, but are not limited
to, magnetic diskettes, magnetic tapes, magnetic disks, optical
disks, CD-ROMs, DVDs, flash memory devices, punched tape or cards,
read-only memory devices, direct access storage devices, gate
arrays, electrostatic memory, and any other like medium.
[0041] "Electropherogram" in reference to the separation of
analytes, molecular tags and the like means a chart, graph, curve,
bar graph, or other representation of signal intensity data versus
a parameter related to the separation process, such as time,
cumulative current, cumulative power and the like, that provides a
readout, or measure, of the number of molecular tags of each type
produced in an assay. A "peak" or a "band" or a "zone" in reference
to an electropherogram means a region where signal intensity values
are high, e.g. relative to background, and correspond to a local
concentration of a separated compound. The value of the time
parameter where a "peak" or "band" occurs is typically referred to
as the "migration time" of that peak. There may be multiple
separation profiles for a single assay, for example, if molecular
tags are labeled with fluorescent dyes and data is collected and
recorded at multiple wavelengths. Thus, molecular tags or
electrophoretic standards that have nearly identical
electrophoretic mobilities may have distinct peaks in
electropherogram data because they are labeled with different dyes.
In one aspect, released molecular tags are separated by differences
in electrophoretic mobility to form an electropherogram wherein
different molecular tags correspond to distinct peaks on the
electropherogram. A measure of the distinctness, or lack of
overlap, of adjacent peaks in an electropherogram is
"electrophoretic resolution," which may be taken as the distance
between adjacent peak maximums divided by four times the larger of
the two standard deviations of the peaks. Preferably, adjacent
peaks have a resolution of at least 1.0, and more preferably, at
least 1.5, and most preferably, at least 2.0. In a given separation
and detection system, the desired resolution may be obtained by
selecting a plurality of molecular tags whose members have
electrophoretic mobilities that differ by at least a peak-resolving
amount, such quantity depending on several factors well known to
those of ordinary skill, including signal detection system, nature
of the fluorescent moieties, the diffusion coefficients of the
tags, the presence or absence of sieving matrices, nature of the
electrophoretic apparatus, e.g. presence or absence of channels,
length of separation channels, and the like. As used herein,
"electropherogram data" means a table, or discrete function,
F(X.sub.i) of signal intensity values for each migration time,
X.sub.i, collected in the electrophoretic separation of molecular
tags. Preferably, electropherogram data comprises fluorescence
intensity values collected by conventional detection systems in a
capillary electrophoresis instrument.
[0042] The term "sample" in the present specification and claims is
used in a broad sense. On the one hand it is meant to include a
specimen or culture (e.g., microbiological cultures), or both
biological and environmental samples used as inputs to an assay. A
sample may include a specimen of synthetic origin. Biological
samples may be animal, including human, fluid, solid (e.g., stool)
or tissue, as well as liquid and solid food and feed products and
ingredients such as dairy items, vegetables, meat and meat
by-products, and waste. Biological samples may include materials
taken from a patient including, but not limited to cultures, blood,
saliva, cerebral spinal fluid, pleural fluid, milk, lymph, sputum,
semen, needle aspirates, and the like. Biological samples may be
obtained from all of the various families of domestic animals, as
well as feral or wild animals, including, but not limited to, such
animals as ungulates, bear, fish, rodents, etc. Environmental
samples include environmental material such as surface matter,
soil, water and industrial samples, as well as samples obtained
from food and dairy processing instruments, apparatus, equipment,
utensils, disposable and non-disposable items. These examples are
not to be construed as limiting the sample types applicable to the
present invention. On the other hand, "sample" is also meant to
refer to a volume of solution analyzed by electrophoresis. Thus,
this volume of solution is placed into a "sample reservoir"
associated with the electrophoretic system and components of the
"sample" are separated.
[0043] A "sieving matrix" or "sieving medium" means an
electrophoresis medium that contains crosslinked or non-crosslinked
polymers, which are effective to retard electrophoretic migration
of charged species through the matrix, wherein such retarding
effect depends at least in part on the molecular shape of the
migrating species. Sieving media are disclosed in Zhu et al, U.S.
Pat. No. 5,089,111; Grossman et al, U.S. Pat. No. 5,126,021;
Madabhushi et al, U.S. Pat. Nos. 5,552,028 and 5,567,292; Shihabi,
Chapter 15, in Landers, editor, Handbook of Capillary
Electrophoresis, Second Edition (CRC Press, Boca Raton, Fla.); and
like references.
[0044] "Specific" or "specificity" in reference to the binding of
one molecule to another molecule, such as a binding compound, or
probe, for a target analyte, means the recognition, contact, and
formation of a stable complex between the probe and target,
together with substantially less recognition, contact, or complex
formation of the probe with other molecules. In one aspect,
"specific" in reference to the binding of a first molecule to a
second molecule means that to the extent the first molecule
recognizes and forms a complex with another molecules in a reaction
or sample, it forms the largest number of the complexes with the
second molecule. In one aspect, this largest number is at least
fifty percent of all such complexes form by the first molecule.
Generally, molecules involved in a specific binding event have
areas on their surfaces or in cavities giving rise to specific
recognition between the molecules binding to each other. Examples
of specific binding include antibody-antigen interactions,
enzyme-substrate interactions, formation of duplexes or triplexes
among polynucleotides and/or oligonucleotides, receptor-ligand
interactions, and the like. As used herein, "contact" in reference
to specificity or specific binding means two molecules are close
enough that weak noncovalent chemical interactions, such as Van der
Waal forces, hydrogen bonding, ionic and hydrophobic interactions,
and the like, dominate the interaction of the molecules. As used
herein, "stable complex" in reference to two or more molecules
means that such molecules form noncovalently linked aggregates,
e.g. by specific binding, that under assay conditions are
thermodynamically more favorable than a non-aggregated state.
[0045] As used herein, the term "spectrally resolvable" in
reference to a plurality of fluorescent labels means that the
fluorescent emission bands of the labels are sufficiently distinct,
i.e. sufficiently non-overlapping, that molecular tags to which the
respective labels are attached can be distinguished on the basis of
the fluorescent signal generated by the respective labels by
standard photodetection systems, e.g. employing a system of band
pass filters and photomultiplier tubes, or the like, as exemplified
by the systems described in U.S. Pat. Nos. 4,230,558; 4,811,218, or
the like, or in Wheeless et al, pgs. 21-76, in Flow Cytometry:
Instrumentation and Data Analysis (Academic Press, New York,
1985).
[0046] "Time", when used in relation to an electropherogram, is
used synonymously with "separation time" meaning the time elapsed
since the initiation of the electrophoretic separation process. The
"migration time" typically refers to the time point at which a
species appears in an electropherogram. For example, "molecular tag
A has a migration time of 10.20 minutes" indicates there is a
signal peak in the electropherogram at a separation time of 10.20
minutes that is due to molecular tag A.
DETAILED DESCRIPTION OF THE INVENTION
[0047] The invention provides systems, methods and
computer-readable products for analyzing one or more compounds by
their electrophoretic properties. In one aspect, such analysis is
carried out by identifying and determining the properties of one or
more peaks in an electropherogram that describes signal intensity
versus cumulative current, or cumulative power, over the course of
a separation. The term "cumulative current" is used synonymously
with "integrated current" or "accumulated charge", and the term
"cumulative power" is used synonymously with "integrated power" or
"accumulated power". Properties of a peak in an electropherogram
include measures of peak area, peak shape, ordinate of the peak's
maximum (referred to herein as the peak "position" or "migration
time"), peak position relative to that of one or more standards
("relative migration time"), and the like.
[0048] The invention operates to correct for fluctuations in
current or power that occur during the normal course of performing
electrophoretic separations and that affect the electropherogram
data. Electropherogram data are transformed from signal versus time
to a new coordinate space of signal versus cumulative current or
power to account for the fluctuations that occur in current or
power during the separation. The use of current or power for the
transformation is determined by the mode of the separation. For a
constant voltage separation, use of the current data is sufficient
for the analysis, whereas when the voltage and current vary, the
power data is used. Electropherograms thus transformed are used for
identifying and determining the properties of the peaks, and
further, the compounds, or equivalently, the analytes, of the
sample.
[0049] In one aspect the invention provides a method of identifying
one or more analytes in a sample using electrophoretic separation
comprising the following steps: (i) applying a potential across a
separation path containing one or more analytes to generate a
current and a power therein and to separate the one or more
analytes so that a first electropherogram of a signal as a function
of time is produced; (ii) integrating the power with respect to
time to provide a cumulative power as a function of time; (iii)
transforming the first electropherogram to a second
electropherogram of the signal as function of the cumulative power;
and (iv) identifying in the second electropherogram peaks that are
correlated with the one or more analytes in the sample. The
potential across a separation path may be constant or it may vary
with time. In one embodiment, the potential across a separation
path may be varied with time so that current in the path or the
power in the path is constant. In another aspect, the method
further comprises the steps of recording the current as a function
of time, recording the potential as a function of time, and
determining the power as a function of time from the recorded
current and voltage. Preferably, a separation path comprises a
capillary tube. During separation of analytes in accordance with
the method of the invention, preferably at least one
electrophoretic mobility standard is provided in the sample,
wherein such standard is used to identify peaks that are correlated
with the one or more analytes of said sample. More preferably, two
mobility standards are provided wherein the mobility of the first
electrophoretic standard is greater than that of any analyte and
the mobility of the second electrophoretic standard is less than
that of any analyte in said sample.
[0050] In one application of the above method, the one or more
analytes of said sample are molecular tags, described more fully
below, wherein each tag has a different electrophoretic mobility.
Preferably, such molecular tags are generated in the sample as the
result of a specific recognition event with at least one type of
biomolecule selected from the group of proteins, antigens,
receptors, DNA and RNA. Usually, the number of molecular tags in
such an embodiment is a plurality in the range of from between 2
and 50.
[0051] In another aspect the invention provides a method of
identifying one or more analytes in a plurality of samples using
electrophoretic separation in the following steps: (i) applying a
potential independently across each of a plurality of separation
paths each containing one or more analytes to generate a current
therein and to separate the one or more analytes in a sample
associated therewith so that for each separation path a first
electropherogram of a signal as a function of time is produced;
(ii) integrating the current in each separation path with respect
to time to provide for each separation path a cumulative current as
a function of time; (iii) transforming each first electropherogram
to a second electropherogram of the signal as function of the
cumulative current for each separation path; and (iv) identifying
in each second electropherogram peaks that are correlated with the
one or more analytes in the sample associated therewith.
[0052] Another embodiment of this aspect of the invention is
carried out in the following steps: (i) applying a potential
jointly across a plurality of separation paths each containing one
or more analytes to generate a current therein and to separate the
one or more analytes in a sample associated therewith so that for
each separation path a first electropherogram of a signal as a
function of time is produced; (ii) integrating the current in each
separation path with respect to time to provide for each separation
path a cumulative current as a function of time; (iii) transforming
each first electropherogram to a second electropherogram of the
signal as a function of the cumulative current for each separation
path; and (iv) identifying in each second electropherogram peaks
that are correlated with the one or more analytes in the sample
associated therewith. As with the aspect of the invention employing
a single separation path, in both embodiments employing pluralities
of separation paths, the potentials across the separation paths may
be constant or may vary with time, one or more standards may be
provided with samples to assist in the identification of analytes,
and separation paths may comprise capillary tubes.
[0053] In another aspect of the invention, a method is provided for
identifying one or more analytes in a sample using electrophoretic
separation comprising the following steps: (i) applying a potential
across a separation path to generate a current therein and to
separate the one or more analytes in the sample, the separation
path having a length, and each of the one or more analytes having
an effective migration distance equal to or less than the length of
the separation path; (ii) recording the current as a function of
time in a series of consecutive segments along the length of the
separation path, each such consecutive segments having a current;
(iii) recording a time series of electropherograms of the signal
intensity associated with the one or more analytes as a function of
the migration distance; (iv) transforming at least one
electropherogram to a second electropherogram of signal intensity
as a function of effective migration distance, wherein the
effective migration distance of an analyte is a function of the
current in each of the consecutive segments of a separation path;
and (v) identifying in such second electropherogram peaks that are
correlated with the one or more analytes in the sample.
[0054] In still another aspect of the invention, a method is
provided for identifying one or more analytes in a sample separated
by electrophoresis to give a first data set of a signal as a
function of time and a data set of the separation path power as a
function of time, the method comprising the steps of: (i)
integrating the separation path power data set with respect to time
to provide a cumulative power as a function of time; (ii)
transforming the first signal data set to a second data set of the
signal as a function of the cumulative power; and (iii) identifying
in the second data set peaks that are correlated with the one or
more analytes in the sample.
[0055] In a further aspect of the invention, a method is provided
for identifying one or more analytes in a sample separated by
electrophoresis to give a first data set of a signal as a function
of time and a data set of the separation path current as a function
of time, the method comprising the steps of: (i) integrating the
separation path current data set with respect to time to provide a
cumulative current as a function of time; (ii) transforming the
first signal data set to a second data set of the signal as a
function of the cumulative current; and (iii) identifying in the
second data set peaks that are correlated with the one or more
analytes in the sample.
[0056] As described more fully below, aspects of the invention may
be implemented using a computer operating under the control of
software instructions recorded on a computer-readable product.
Accordingly, an aspect of the invention is a computer-readable
product embodying a program for execution by a computer to identify
one or more analytes in an electrophoretic separation by
determining peak locations in a transformed electropherogram and
correlating such peak locations with each of the one or more
analytes, the program comprising instructions for carrying out the
following steps: (i) reading a first electropherogram data set
based on an analyte signal as a function of separation time from a
data storage medium; (ii) reading a data set of power as a function
of separation time from a data storage medium; (iii) determining a
data set of cumulative power as a function of separation time; (iv)
transforming the first electropherogram data set to a second
electropherogram data set of the analyte signal as a function of
the cumulative power; (v) identifying peak locations in the second
electropherogram; and (vi) correlating the identified peak
locations with each of the one or more analytes. Preferably, the
step of reading a data set of power as a function of separation
time comprises: (i) reading a data set of current as a function of
separation time from a data storage medium; (ii) reading a data set
of potential as a function of separation time from a data storage
medium; and (iii) determining using the current and the potential
data sets, a data set of power as a function of separation
time.
Methods and Instrumentation for Electrophoretic Separation
[0057] Methods for electrophoresis of are well known and there is
abundant guidance for one of ordinary skill in the art to make
design choices for forming and separating particular pluralities of
compounds. The following are exemplary references on
electrophoresis: Krylov et al, Anal. Chem., 72: 111R-128R (2000);
P. D. Grossman and J. C. Colburn, Capillary Electrophoresis: Theory
and Practice, Academic Press, Inc., NY (1992); U.S. Pat. Nos.
5,374,527; 5,624,800; 5,552,028; ABI PRISM 377 DNA Sequencer User's
Manual, Rev. A, January 1995, Chapter 2 (Applied Biosystems, Foster
City, Calif.); and the like. In one aspect, one or more analytes
are separated by capillary electrophoresis and the resulting
electropherogram transformed for analysis. Design choices within
the purview of those of ordinary skill include but are not limited
to selection of instrumentation from several commercially available
models, selection of operating conditions including separation
media type and concentration, pH, desired separation time,
temperature, voltage, capillary type and dimensions, detection
mode, the number of analytes to be separated, and the like.
[0058] In one aspect of the invention, during or after
electrophoretic separation, the analytes are detected or identified
by recording fluorescence signals and migration times (or migration
distances) of the separated compounds, or by constructing a chart
of relative fluorescence as a function of time or order of
migration of the analytes (e.g., as an electropherogram). To
perform such detection, the analytes can be illuminated by standard
means, e.g. a high intensity mercury vapor lamp, a laser, or the
like. Typically, the analytes are illuminated by laser light
generated by a He--Ne gas laser or a solid-state diode laser. The
fluorescence signals can then be detected by a light-sensitive
detector, e.g., a photomultiplier tube, a charged-coupled device,
or the like. Exemplary electrophoresis detection systems are
described elsewhere, e.g., U.S. Pat. Nos. 5,543,026; 5,274,240;
4,879,012; 5,091,652; 6,142,162; or the like. In another aspect,
analytes may be detected electrochemically detected, e.g. as
described in U.S. Pat. No. 6,045,676.
[0059] Electrophoretic separation involves the migration and
separation of molecules in an electric field based on differences
in mobility. Various forms of electrophoretic separation include,
by way of example and not limitation, free zone electrophoresis,
gel electrophoresis, isoelectric focusing, isotachophoresis,
capillary electrochromatography, and micellar electrokinetic
chromatography. Capillary electrophoresis involves
electroseparation, preferably by electrokinetic flow, including
electrophoretic, dielectrophoretic and/or electroosmotic flow,
conducted in a tube or channel of from about 1 to about 200
micrometers, usually, from about 10 to about 100 micrometers
cross-sectional dimensions. The capillary may be a long independent
capillary tube or a channel in a wafer or film comprised of
silicon, quartz, glass or plastic.
[0060] In capillary electroseparation, an aliquot of the reaction
mixture containing the analytes is subjected to electroseparation
by introducing the aliquot into an electroseparation channel that
may be part of, or linked to, a capillary device in which an assay,
an amplification reaction or other reactions are performed. An
electric potential is then applied to the electrically conductive
medium contained within the channel to cause migration of the
components within the combination. Generally, the electric
potential applied is sufficient to achieve electroseparation of the
desired components according to practices well known in the art.
One skilled in the art will be capable of determining the suitable
electric potentials for a given set of reagents, compounds or
analytes and/or the nature of the samples, the nature of the
reaction medium and so forth. The parameters for the
electroseparation including those for the medium and the electric
potential are usually optimized to achieve maximum separation of
the desired components. This may be achieved empirically and is
well within the purview of the skilled artisan.
[0061] Detection may be by any of the known methods associated with
the analysis of capillary electrophoresis columns including the
methods shown in U.S. Pat. Nos. 5,560,811 (column 11, lines 19-30),
4,675,300, 4,274,240 and 5,324,401, the relevant disclosures of
which are incorporated herein by reference. Those skilled in the
electrophoresis arts will recognize a wide range of electric
potentials or field strengths may be used, for example, fields of
10 to 1000 V/cm are used with about 200 to about 600 V/cm being
more typical. The upper voltage limit for commercial systems is
about 30 kV, with a capillary length of about 40 to about 60 cm,
giving a maximum field of about 600 V/cm. For DNA, typically the
capillary is coated to reduce electroosmotic flow, and the
injection end of the capillary is maintained at a negative
potential.
[0062] For ease of detection, the entire apparatus may be
fabricated from a plastic material that is optically transparent,
which generally allows light of wavelengths ranging from about 180
to about 1500 nm, usually about 220 to about 800 nm, more usually
about 450 to about 700 nm, to have low transmission losses.
Suitable materials include fused silica, plastics, quartz, glass,
and so forth.
[0063] FIG. 1A is a schematic illustration of an exemplary
capillary electrophoresis system 10 for performing the method of
the present invention. In the figure, the separation path is
capillary tube 12 containing separation medium 13, and which spans
between a cathodic reservoir 14 and an anodic reservoir 16, both of
which contain a conducting electrolyte medium. Generally, a
"separation path" is a geometrically-defined route within which the
separation medium is confined and along which a potential gradient
is established. Depending on the type of electrophoresis instrument
or equipment, a separation path is variously referred to as a
"channel", "capillary", or "lane", the latter being the common
nomenclature for conventional molecular biology slab gels. A sample
reservoir 18 and reservoir 14 are interchangeably contacted with
capillary tube 12 to provide for introduction of the sample, which
may be accomplished electrokinetically or pneumatically. The
relationship between the anodic and cathodic reservoirs in FIG. 1A
may be reversed, according to the nature of the analytes being
analyzed. As illustrated here, the setup is appropriate for the
analysis of anionic analytes, which are drawn into the capillary
tube 12 containing separation medium 13 and past detector 20,
towards the anodic reservoir 16. Power supply 22 applies a
potential across the separation path via the cathodic 24 and anodic
26 electrodes that are contacted to reservoirs 14 and 16 so that a
potential gradient, equivalently an electrical field, collinear
with the separation path is established. The polarity of the
connection of the electrodes to the reservoirs determines the
direction of the potential gradient and the ion movement and thus
whether anionic or cationic analytes are analyzed at detector 20. A
current measuring device 28 and voltage measuring device 30 for
measuring, respectively, the electrophoretic current in the
separation path and the voltage, or equivalently, the potential
across the separation path may also be associated with system
10.
[0064] The system 10 is operated under the control of computer
processor 32. The processor 32 communicates with power supply 22,
current measuring device 28 and voltage measuring device 30 via
cable 34, and communicates with detector 20 via cable 36. The
cables 34 and 36 may provide for one-way or two-way communication.
In the former case of one way communication, the data measured by
devices 28 and 30, and detector 20 are typically transferred to the
processor wherein the data may be manipulated, stored, displayed,
further transmitted to another computer or likewise treated as data
sets. Where the cables act as a two-way data bus, signals for
powering, controlling, adjusting or otherwise tuning the voltage,
current and power of the electrophoretic separation and the
detector function may be sent from the processor 32 to the various
components. The power supply 22 itself may also function to control
and adjust the power, voltage and current applied during the
electrophoretic separation. For example, power supplies that
operate in constant voltage, constant current, or constant power
modes are available.
[0065] As noted, several manufacturers have available automated
instruments for capillary electrophoresis that may be used in
accordance with the invention. Some instruments provide only one
capillary tube, while others contain a bundled array of tubes,
varying from 4 to 16 to 96 or even 256 tubes, for what is referred
to as capillary array electrophoresis.
[0066] The operation is exemplified with a sample that is a
particular assay mixture, although it should be understood that any
sample type or mixture of compounds that is used in the art of
electrophoretic separations is within the intended scope of the
invention. The assay mixture, which as noted below contains one or
more targets, one or more tagged protein or DNA probes, and
optionally, at least one electrophoretic standard, is placed in
sample reservoir 18. The assay reaction, involving initial probe
binding to target(s) followed by the release of molecular tags,
which in this example are the analytes, may be carried out in
sample reservoir 18, or alternatively, the assay reactions can be
carried out in another reaction vessel, with the reacted sample
components then added to the sample reservoir.
[0067] The sample reservoir 18 is brought into contact with
capillary tube 12 and the sample is injected into the separation
medium 13 in the tube either by application of a potential or
pressure. Once injected, the tube 12 is contacted with reservoir
14, the power supply 22 applies a voltage via electrodes 24 and 26
to the reservoirs 14 and 16 to cause the formation of a potential
gradient along the separation path 12 and thus the migration of
charged components of the sample through the separation medium 13.
As analytes move past detector 20, a signal indicating their
presence and amount is recorded as a function of the time by
processor 32 to form a first electropherogram. Also during the
separation the current or the power in the separation path is
recorded as a function of time. As disclosed more fully below, the
current or power data set is integrated to provide the cumulative
current or power, the first electropherogram is transformed to a
second electropherogram of signal as a function of the cumulative
current or power, and the peaks are identified by correlation to
the analytes in the sample.
[0068] FIG. 1B is a schematic illustration of a slab gel
electrophoresis system 50 useful for performing the invention,
wherein like-numbered components with FIG. 1A perform the same
function as described above. The slab gel comprises separation
paths 52a-52e comprising separation medium 53. Although the
separation paths in a gel are in fluidic and electrical
communication with one another, the migrating samples move
substantially in a direct line along the potential gradient and
remain substantially isolated from one another, and thus each
sample is said to be confined with a lane. The slab gel may be a
free-standing gel such as is commonly performed in the art for
agarose gels, or the gel may be supported between two, narrowly
spaced plates such as is known in the art for polyacrylamide gels.
Slab gels are variously oriented horizontally or vertically,
depending on the type of gel and separation being performed. See,
for example, U.S. Pat. Nos. 4,830,725 and 4,773,984, respectively,
for examples of each gel type.
[0069] Generally, wells 58a-58e are preformed in the separation
medium and are the means by which samples are introduced to the
separation medium in each lane 52a-52e. For illustrative purposes
only a limited number of wells and separation paths 52a-52e are
depicted, however the number of wells, the width and the spacing of
the wells will be varied according to the number of samples,
desired throughput, scale, resolution, power supply capability and
the like as is commonly known in the art. It is appreciated that in
slab gels, the separation paths for each sample are not isolated
from one another as is the case for capillary tube arrays, but
rather these separation paths are in fluid and electrical
communication. Nonetheless, as is known by those skilled in the
art, the samples are maintained within distinct lanes and the
detection process generates distinct electropherograms.
[0070] The detector 60 measures the signal associated with the
analytes as function of the separation time in each lane. The
detector 60 may be responsive to any of the typical signals used in
conjunction with gel electrophoresis, especially the emitted
visible or infrared light produced by fluorophores. For convenience
the detector may only respond to a small area, i.e. one lane or a
fraction thereof, and be periodically scanned across all the lanes
in order to measure the signal for each lane, e.g. as disclosed by
Hunkapiller et al. in U.S. Pat. No. 4,811,218.
[0071] In operation, samples are transferred to the sample
reservoirs 58a-e. A power supply 22 applies a voltage via
electrodes 24 and 26 to the reservoirs 14 and 16 to establish a
potential gradient along the separation paths 52a-52e in the gel,
and thus cause electrophoretic migration of the charged components
of the sample through the separation medium 53. Detector 20
monitors a signal indicating the presence and the amount of each
analyte in each separation path, which is recorded by processor 32
to form a first electropherogram for each separation path. Also
during the separation the current or the power in the separation
paths is recorded as a function of time. As disclosed more fully
below, the current or power data set is integrated to provide the
cumulative current or power, the first electropherograms are
transformed to second electropherograms of signal as a function of
the cumulative current or power, and the peaks are identified by
correlation to the analytes in the samples.
[0072] In another aspect of the invention, the electrophoretic
separation is carried out in a microfluidics device, as illustrated
diagrammatically in FIGS. 1C-1E. Microfluidics devices are
described in a number of domestic and foreign Letters Patent and
published patent applications. See, for example, U.S. Pat. Nos.
5,750,015; 5,900,130; 6,007,690; and WO 98/45693; WO 99/19717 and
WO 99/15876. Conveniently, an aliquot, generally not more than
about 5 .mu.L, is transferred to the sample reservoir of a
microfluidics device, either directly through electrophoretic or
pneumatic injection into an integrated system or by syringe,
capillary or the like. The conditions under which the separation is
performed are conventional and will vary with the nature of the
products.
[0073] By way of illustration, FIGS. 1C-1E show a microchannel
network 100 in a microfluidics device of the type detailed in the
application noted above, for sample loading and electrophoretic
separation of a sample of probes and tags produced in the assay
above. Briefly, the network includes a main separation channel 102
terminating at upstream and downstream reservoirs 104, 106,
respectively. The main channel is intersected at offset axial
positions by a first side channel 108 that terminates at a
reservoir 110, and a second side channel 112 that terminates at a
reservoir 114. The offset between the two side channels forms a
sample-loading zone 116 within the main channel.
[0074] In operation, a sample or an assay mixture is placed in
sample reservoir 110, illustrated in FIG. 1C. Assay reactions may
be carried out in sample reservoir 110, or alternatively, the assay
reactions can be carried out in another reaction vessel, with the
reacted sample components then added to the sample reservoir.
[0075] To load the analytes into the sample-loading zone, an
electric field is applied across reservoirs 110, 114, in the
direction indicated in FIG. 1D, wherein negatively charged analytes
are drawn from reservoir 110 into loading zone 116, while uncharged
or positively charged sample components remain in the sample
reservoir. The analytes in the loading zone can now be separated by
conventional capillary electrophoresis, by applying an electric
filed across reservoirs 104, 106, in the direction indicated in
FIG. 1E.
[0076] As analytes move past a detector, a signal indicating their
presence and amount is recorded as a function of the time by a
processor to form a first electropherogram. Also during the
separation the current or the power in the separation path is
recorded as a function of time. As disclosed more fully below, the
current or power data set is integrated to provide the cumulative
current or power, the first electropherogram is transformed to a
second electropherogram of signal as a function of the cumulative
current or power, and the peaks are identified by correlation to
the analytes in the sample.
[0077] Other operating methods and designs for microfluidics CE
devices are known in the art, such as described in U.S. Pat. Nos.
5,858,195; 6,001,229, 6,010,607, 6,110,332; 6,143,152; or the
commercially available Bioanalyzer 2100 (Agilent Technologies,
Santa Clara, Calif.), may also be used in conjunction with the
present invention without limitation.
Measuring Current Voltage and Power
[0078] As mentioned above, an object of the invention is to provide
improved analyte identification by correcting for fluctuations in
current or power within separation paths in an electrophoretic
separation. The mobility (v) of a charged species in an electric
field is given by the equation:
v=(qE)/6.pi.r.eta.
[0079] where q is the charge of the species, E is the potential
field strength (i.e. (V/d), the voltage applied (V) divided by the
distance (d) over which it is applied), r is proportional to the
size of the species and .eta. is the viscosity of the separation
medium (Physical Chemistry, P. W. Atkins and J. de Paula, 7.sup.th
ed., 2001). When applied in the context of electrophoresis, the
mobility can be used to determine an expected migration time of the
charged species along a separation path from e.g., the inlet to the
detector. However, this requires several assumptions be made, such
as that the voltage applied during the separation and the viscosity
of the separation medium, which is a strong function of the
temperature, are constant. Electrophoresis instruments are
generally designed and operated in a manner that controls or
minimizes these variations, e.g. by operating at constant voltage
and actively regulating the temperature, in order to provide
consistent and reproducible performance.
[0080] In most cases though the design is not able to address all
the causes of variation and differences in the data from run to run
persist. For example, in instruments with multiple capillaries the
potential gradient established in each capillary will differ to the
extent that the capillaries differ in length. Thus, to minimize
run-to-run variations, practitioners develop standard methods and
adhere to standard protocols. However even in this environment,
samples, especially those derived from biological sources, may have
different compositions of ions and other charged species.
Differences in the ionic content of samples will manifest itself in
different behavior during sample injection into the capillary.
Consequently, different sample plug injections will yield different
local conditions in the separation path of the electrophoretic
analysis.
[0081] Electrophoretic analysis generally consists of effecting a
separation using constant voltage while recording the signal as a
function of time (or distance). However, even if a constant voltage
is maintained, the potential gradient in the separation path
typically varies both from path to path, as well as along and
within the length of each path, giving rise to non-uniform
migratory trajectories and varying current profiles.
Electrophoretic mobility standards are often used as a means to
correct for run-to-run variations by analyzing the mobility of the
analytes as a ratio with respect to the mobility of the standards.
This provides a first-order correction in some cases, but there are
still occasions in which the variation in the separation
performance is non-linear.
[0082] The information provided by the current or the power in the
separation path as a function of the separation time is employed in
the present invention to further incorporate the conditions present
during the separation into the information content of the
electropherogram. By measuring the current and voltage present in
the separation path, the current or power profile during the
separation can be used to correct the electropherogram graph. In
cases where the separation is performed at constant voltage,
monitoring and recording the current provides sufficient
information regarding the variation during the run. Conversely, if
the separation is run at constant current, then monitoring and
recording the voltage provides the necessary information regarding
the variation during the run. In some cases neither the voltage nor
current is strictly regulated, in which case both parameters are
monitored and recorded in order that the power as a function of
time is known. Power meters may be also be used to monitor and
record the power, although fundamentally the measuring device
operates by determining separately the current and the voltage. In
some protocols, a series of different constant voltage or constant
current conditions are established in the separation path. In yet
some other protocols, a known, varying profile of voltage or
current is established in the separation path. In these other
protocols there is the need to monitor and record both the voltage
and current as a function of time in order to know the power
profile during the separation.
[0083] Current in a circuit is measured by an ammeter, which is
typically constructed of a circuit comprising a shunt resistor. The
voltage difference between two points in a circuit is measured by a
voltmeter, which is typically constructed of a circuit comprising a
series resistor. Digital ammeters and digital voltammeters that
also provide autoscaling and data logging capability are
commercially available (from e.g. Fluke Corporation, Everett,
Wash., or Agilent Technologies, Santa Clara, Calif.). Furthermore
the circuit designs of ammeters and voltammeters are well known to
those skilled in the electronic arts. Power in a circuit a product
of the current multiplied by the voltage. The power may be measured
by a wattmeter, or a dynamometer. More typically, the current and
voltage are measured separately and multiplied together. The
measurements and recorded data sets may be analog in format, but
for ease of further manipulation, calculation and storage, data in
a digital format are preferred.
[0084] Current, voltage and power measurement capability are often
provided in power supply instruments. Furthermore, automated
electrophoresis instruments also generally comprise current and
voltage measurement capabilities. For example, the MegaBACE 1000
capillary array electrophoresis instrument (Amersham Biosciences,
Piscataway, N.J.) provides records of current and voltage
measurements for each capillary, and the ABI 3100 (Applied
Biosystems, Foster City, Calif.) provides records of current and
voltage measurements for the collective array of capillaries.
[0085] In the present invention the current, voltage or power is
measured periodically during the separation process in order to
provide a data set of the measurement as a function of the
separation time. The choice of the parameters of current, voltage
or power that are to be measured is discussed in more detail below.
The measurements are used to provide a data set of the current,
voltage or power at time points corresponding to the time points of
the signal measurement provided by the detector. It is noted that
the frequency of the signal measurement is set according to the
requirements of forming an electropherogram with sufficient
resolution of the peaks.
[0086] Preferably, the frequency of measurement of current, voltage
or power is approximately the same as the sampling rate of the
detector in recording the signal associated with the analytes,
although the frequency may be higher. If the frequency of the
current, voltage or power measurement is less than half the
frequency of the detector measurement (i.e. the measurement is made
once in a period of two or more detector readings), interpolation
of the current, voltage or power data may be used to generate data
values corresponding to the time point of each detector reading.
Likewise if the value of the time points for the current, voltage
or power measurements are different from those of the signal data
set, again interpolation may be used to generate data values at the
corresponding time points. Either the signal data set or the
current, voltage or power data sets may be interpolated to provide
data at time points corresponding to those of the other group.
Electrophoretic Analysis with Transformed Electropherograms
[0087] The invention provides a means for identifying analytes in a
sample by electrophoresis, particularly for transforming the
obtained electropherogram, i.e. a first electropherogram, to a
representation of the signal as a function of the electrical
parameters of the separation process, whereupon the transformed
electropherogram, i.e. a second electropherogram, is used as the
basis for peak identification and correlation with the expected
migration time of the analytes.
[0088] Referring to FIG. 2A, the steps, according to one embodiment
of the present invention, of a method for identifying one or more
analytes in a sample by electrophoresis are enumerated. The method
comprises first applying a potential across a separation path (150)
to generate a current therein and to separate the one or more
analytes in the sample by electrophoresis to produce an
electropherogram of a signal as a function of time. Suitable
electrophoresis systems are exemplified in FIGS. 1A-1E. In a
preferred embodiment, the potential applied across the separation
path is constant, using for example a constant voltage power
supply.
[0089] The next step is integrating the current with respect to
time (152) to provide the cumulative current as a function of time.
As noted, it is preferred that the data be in a digital format,
i.e. that data sets are series of discrete values. Thus, the
preferred integration method uses numerical analysis. Examples of
numerical analytical integration methods are described in Numerical
Computation 2: Methods, Software and Analysis, by C. W. Ueberhuber
(Springer-Verlag, 1997). One preferred method of integration to
determine the cumulative current up to each time point is the
following summation: 1 I c ( t ) = I 0 + i = 1 t I i ; for each t ,
from t = 1 to t = T
[0090] where I.sub.c is the cumulative current, t is each sampled
time point, I.sub.0 is the current at t=0, I.sub.i is the current
sampled at the i.sup.th time point, and T is the total separation
time, to generate a data set of the series of I.sub.c as a function
of time.
[0091] The next step is transforming the electropherogram to a
second electropherogram (154) of the signal as a function of the
cumulative current. The method of the invention calls for mapping
the electropherogram to a new coordinate system based on the
electrical characteristics, the current or the power, of the
separation. The two measured quantities, the signal and the
cumulative current, are explicit functions of an independent
variable, time. This relationship defines parametric equations. The
transformation is carried out by forming ordered pairs of values of
the signal and cumulative current for each time point in the
separation. Thus, the second electropherogram represents the signal
as a function of the cumulative current and may be graphed and
analyzed in manner analogous to signal versus time plots.
[0092] The final step in the method is identifying (156) in the
second electropherogram peaks that are correlated with the one or
more analytes in the sample. Using the transformed, or second,
electropherogram as the basis for the analysis, the procedure for
peak identification is fully described in a following section.
[0093] In another embodiment, the potential applied across the
separation path varies with time. In this case, the current is no
longer sufficient for the transformation, and the potential must
also be used. An appropriate integration method is the following
summation: 2 V I c ( t ) = V 0 I 0 + i = 1 t V i I i ; for each t ,
from t = 1 to t = T
[0094] where V.multidot.I.sub.c is the cumulative
voltage.multidot.current product, t is each sampled time point,
V.sub.0 is the potential and I.sub.0 is the current at t=0, V.sub.t
is the potential and I.sub.i is the current sampled at the i.sup.th
time point, and T is the total separation time, to generate a data
set of the series of V.multidot.I.sub.c as a function of time. In
this embodiment the first electropherogram is transformed to a
second electropherogram of the signal as a function of the current
and potential.
[0095] In another embodiment, the method comprises applying a
potential across a separation path to generate a current and a
power therein, and then integrating the power with respect to time
to provide the cumulative power as a function of time. Here, the
appropriate integration method is: 3 P c ( t ) = P 0 + i = 1 t P i
; for each t , from t = 1 to t = T
[0096] where P.sub.c is the cumulative power, t is each sampled
time point, P.sub.0 is the power at t=0, P.sub.i is the power
sampled at the i.sup.th time point, and T is the total separation
time, to generate a data set of the series of P.sub.c as a function
of time. In preferred embodiments the power is obtained as the
product of the current multiplied by the potential, which are each
known by independent measurement, and thus the appropriate terms in
the summation are explicitly given in the previous equation. The
transformation is then carried out by forming ordered pairs of
values of the signal and cumulative power for each time point.
[0097] In yet another embodiment, the invention provides a method
of identifying one or more analytes in a sample separated by
electrophoresis to give a first data set of a signal as a function
of time and a data set of the separation path current as a function
of time. The method comprises integrating the separation path
current data set to provide a cumulative current as a function of
time, the preferred method of integration being numerical analysis
techniques. The next step is transforming the first signal data set
to a second data set of the signal as a function of the cumulative
current, as described earlier via the parametric relationship, and
then identifying in the second data set peaks that are correlated
with the one or more analytes in the sample.
[0098] Another aspect of the present invention is a method of
identifying one or more molecular tags in a sample using
electrophoretic separation. At least one electrophoretic mobility
standard is added to the sample prior to separation. Preferably,
two mobility standards are added, more preferably wherein one has a
greater mobility and the other has a lesser mobility than that of
any analyte in the sample. The molecular tags are present in the
electrophoresis sample as result of an assay for biomolecules, such
as proteins, antigens, antibodies, receptors, or nucleic acids
(DNA, RNA and the like) in a biological sample, examples of which
are further described below. Provided such a sample of molecular
tags, preferably a plurality of tags which may number from 2 to 20
or even as many as 50, the above described methods are followed to
achieve the identifying of the analytes.
[0099] In some methods of electrophoresis, the result of the
separation is analyzed on the basis of the migration distance, that
is, the distance that each analyte has migrated during the
separation. Where local variations in the separation conditions
have affected the local mobility of the analytes differentially,
the present invention contemplates methods for correcting for these
fluctuations and differential perturbations.
[0100] The first step of a method for identifying analytes in an
electrophoretic separation using migration distance is applying a
potential across the length of a separation path to generate a
current therein and to separate the one or more analytes in each
sample by electrophoresis. During the separation, the current is
recorded as a function of time in a series of consecutive segments
of the separation path. The separation path is divided into
segments and each segment is provided with means for measuring and
recording the current in that segment as a function of time. For
example, a series of electrodes fabricated on the surface defining
the separation path can define the series of segments. The
electrodes are used as probes, and, with the use of external
circuitry such as is used in digital multimeters for the diagnosis
of electronic circuits, provide measurements of the resistivity,
potential difference and current between any two electrodes
defining a segment.
[0101] The method also calls for recording a time series of
electropherograms of the signal intensity associated with the
analyte as a function of the migration distance. The time series of
electropherograms are recorded at about the same frequency as the
current measurements in each segment. The data sets of the current
as a function of location and the signal vs. distance are the basis
for transforming at least one electropherogram to represent the
signal intensity as a function of the effective migration distance,
wherein the effective migration distance is a function of the
current experienced by each peak in each separation path segment.
Finally, the transformed electropherogram is used to identify in
the at least one electropherogram peaks that are correlated with
the one or more analytes in the sample.
[0102] The invention also provides systems for carrying out the
above methods. It is contemplated that in addition to the single
channel capillary electrophoresis apparatus exemplified in FIG. 1A,
multichannel CE instruments may also be used. Two such systems are
illustrated in FIGS. 2B and 2C, where for purposes of clarity
three-channel CE systems are shown. As noted, there are several
types of capillary array electrophoresis instruments that have from
4 to 256 capillaries, as well as planar CE devices that accommodate
e.g. 12 samples. Comparing FIGS. 2B and 2C with FIG. 1A,
like-numbered components perform the same function and operation as
described previously. In a multichannel system there are design
choices to be made in the construction of an instrument. FIG. 2B
illustrates a system 160 with independent capillaries. The same
potential is applied in parallel by power supply 22 across each of
the three separation paths 12a-c, but the current generated in each
path is independent of the others. Thus the current or power may be
separately monitored and recorded for each path, for example by
current measuring devices 28a-c. The signal associated with the
analytes in each separation path is measured by detector 20. In
this manner, the analysis and identification of the analytes in
each sample may be performed in the same manner as described above
since the same information is independently available for each
sample. Reservoirs 16a-c may be combined to be in fluid
communication without loss of function in the illustrated scenario
of FIG. 2B.
[0103] In FIG. 2C, a system 170 with the several capillaries
terminating at each end in common reservoirs 14 and 16 is
illustrated. The difference between this design and that of FIG. 2B
is that though the potential may applied in parallel across all the
separation paths 12a-c, the current or power can no longer be
independently measured for each separation path. This situation
also obtained in the slab gel electrophoresis system of FIG. 1B. In
this case the current for each path is determined to be the
prorated share of current for each separation path based on the
known geometry, resistivity, temperature, and other physical
characteristic of each path. In a preferred embodiment, the
prorated share of current is determined solely by the relative
geometric cross-section of each separation path. For example, in
system 170, assuming the three capillary tubes 12a-c have identical
cross-sectional areas, the prorated share of current in each path
is the total current, as measured by ammeter 28, divided by three.
In this manner, the analysis and identification of the analytes in
each sample may be performed as previously described using the
signal recorded by detector 20, the applied potential and the
prorated current for each separation path.
[0104] In one embodiment of a system for performing the invention,
the system comprises, with reference to FIG. 1A, a separation path
12, a voltage source 22 for applying a potential across the
separation path 12 wherein a current is generated, a detector 20
positioned along the separation path 12 for recording a first
electropherogram of the signal as a function of time, and a
processor 32 comprising software for integrating the current to
provide the cumulative current as a function of time, transforming
the first electropherogram to a second electropherogram of the
signal as a function of the cumulative current, and identifying in
the second electropherogram peaks that are correlated with the
analytes in the sample. In a preferred embodiment the voltage
source applies a constant potential.
[0105] In another embodiment, the potential and current vary during
the separation, in which case the power in the separation path is
integrated, and the second electropherogram is a function of the
cumulative power.
Computer System and Programs
[0106] A computer preferably performs steps of transforming the
electropherogram and the method of identifying peaks in
electropherogram data described above. In one embodiment, a
computer comprises a processing unit, memory, I/O device, and
associated address/data bus structures for communicating
information therebetween. The processing unit may be a conventional
microprocessor driven by an appropriate operating system, including
RISC and CISC processors, a dedicated microprocessor using embedded
firmware, or a customized digital signal processing circuit (DSP),
which is dedicated to the specific processing tasks of the method.
The memory may be within the microprocessor, i.e. level 1 cache,
fast S-RAM, i.e. level 2 cache, D-RAM, flash, or disk, either
optical or magnetic. The I/O device may be any device capable of
transmitting information between the computer and the user, e.g. a
keyboard, mouse, network card, or the like. The address/data bus
may be a PCI bus, NU bus, ISA, or any other like bus structure.
When the computer performs the method of the invention, the
above-described method steps are embodied in a program stored in or
on a computer-readable product. Such computer-readable product may
also include programs for graphical user interfaces and programs to
change settings on electrophoresis systems or data collection
devices.
[0107] The invention also provides a computer-readable product for
identifying one or more analytes by determining peak locations in a
transformed electropherogram and correlating the peaks with the
analytes. In one embodiment, the product comprises the listed
instructions of FIG. 2D. A first electropherogram data set of a
signal as a function of separation time (180) and a data set of
current as function of time (182) are read into memory. The current
data set is integrated using numerical methods to provide a data
set of the cumulative current as a function of time (184), which
may also be stored in memory. As discussed above, in some cases a
data set of power as a function of time is preferred over the data
set of current. The first electropherogram is transformed as
discussed above to a second electropherogram of the signal as a
function of the cumulative current (186). In the second
electropherogram, the peak locations are identified (188), as
discussed in the following section, and the peak locations are
correlated with the analytes.
[0108] Recognizing that in some cases peak identification and
correlation with analytes are performed manually, or by visual
inspection, another embodiment of the invention provides a
computer-readable product for transforming electropherograms. In a
manner similar to that described in conjunction with FIG. 2D, a
first electropherogram data set of a signal as a function of time
is read into memory, and a data set of either a current or power as
a function of time is also read into memory. The data set of
current or power is integrated using numerical methods to provide
the cumulative current or power as a function of time. Finally the
first electropherogram is transformed to a second electropherogram
representing the signal as a function of the cumulative current or
power, to provide a transformed electropherogram for further use or
inspection.
Peak Identification From Electropherogram Data
[0109] In the following discussion, the electropherograms are
stated to be representing the signal as a function of time for
convenience. In the present invention, the method of analysis
involves transforming electropherograms from signal versus time to
signal versus cumulative current or cumulative power to correct the
data for variations in the separation conditions over time. These
transformations may be regarded as mapping the electropherogram
onto a function of modified time. Accordingly, in the following
discussion the terms time, migration time, relative migration time
and the like should be viewed as being related to the modified
time, with the relationship between time and modified time being
determined by the method used for transforming the first
electropherogram to a second electropherogram.
[0110] Also in the following discussion, the analytes are
exemplified as being "molecular tags", however the discussion
should be understood to be generally applicable to all types of
analytes, compounds and species that are separated and analyzed in
the arts of electrophoretic separations. It is commonly understood
in the art that the methods of analysis of the results of a
separation are independent of the particular composition of the
sample being analyzed.
[0111] A typical electropherogram (200) displaying electropherogram
data is illustrated in FIG. 3A. Several peaks are shown, including
a first electrophoretic standard (202) ("std.sub.1"), peaks
corresponding to molecular tags mT.sub.1 through mT.sub.6, and a
second electrophoretic standard 9204 ("std.sub.2"). Factors that
complicate the identification, or correlation, of peaks with
molecular tags include noise (205) that may be time dependent,
variability between adjacent peaks, or stretching or compressions
(208), elevation or variability in the "baseline" signal (206), and
the like. As explained more fully below, an object of the present
invention is to provide methods for accurately correlating peaks in
electropherogram data with molecular tags in view of the
above-mentioned distortions in the data. As illustrated in FIG. 3B,
in one aspect, the invention provides measures of peak locations
relative to the positions of one or more electrophoretic standards.
In particular, a migration time T.sub.3 (252) for a molecular tag,
"mT.sub.3", is provided as the following ratio:
T.sub.3=(t.sub.3-T.sub.s1)/(T.sub.s2-T.sub.s1)
[0112] where t3 is the observed migration time and T.sub.s1 and
T.sub.s2 are the migration times of electrophoretic standards (202)
and (204), respectively.
[0113] The method of correlating peaks in electropherogram data
with molecular tags follows the general steps in FIG. 3C. After
electropherogram data is read (290) by a processing unit, peak
locations are identified (292) and peak sizes are determined (294).
Finally, all or a subset of identified peaks are correlated (296)
with molecular tags used in the assay. Preferably, peak size is
correlated to the amount of analyte in a sample. A variety of
measures may be used for peak size, including peak height, peak
area, or the like. Preferably, peak area is used as a measure of
peak size. Peak area may be estimated is a variety of ways,
including taking the product of peak height and peak width at half
maximum height, curve fitting, numerical integration of peak areas,
and the like.
[0114] In one aspect of the invention, two electrophoretic
standards are employed, a first electrophoretic standard, e.g.
(202) in FIG. 3A, and a second electrophoretic standard, e.g. (204)
in FIG. 3A. All other molecular tags used in an assay are selected
so that their peaks in electropherogram data falls between the
first electrophoretic standard and the second electrophoretic
standard, e.g. as illustrated by molecular tags, "mT.sub.1",
"mT.sub.2", "mT.sub.3", "mT.sub.4", "mT.sub.5", and "mT.sub.6",
shown in FIG. 3A. In other embodiments, more than two
electrophoretic standards may be used and the locations of the
standards may be among the peaks corresponding to molecular tags,
and not necessarily before and after the locations of such
peaks.
[0115] Another aspect of the invention makes use of the fact that
molecular tags are designed to have either predetermined
electrophoretic mobilities and optical properties. If sufficient
numbers of a particular tag are released in an assay, then that
molecular tag may itself serve as an electrophoretic standard for
identification of subsequent peaks. This is advantageous because
the closer the reference peak or standard is to a peak whose
location is being determined, the more accurate the value for the
peak location. As used herein, the term "qualified peak" refers to
a peak in electropherogram data that is correlated to a particular
molecular tag and that fulfills predetermined criteria for use as
an electrophoretic standard. Such criteria may include a measure
for peak signal-to-noise ratio, absolute peak height, peak width,
or the like. Preferably, a peak is a qualified peak if the peak
signal-to-noise ratio is greater than or equal to 1.5; and more
preferably, 2.0; and still more preferably, 2.5. In this
embodiment, the accuracy of peak identification may vary according
to the presence or absence of analytes in a sample because all,
some, or none of the molecular tags may be released in detectable
amounts, thereby giving rise to a greater or lesser number of
available standards.
[0116] In another aspect of the invention, illustrated in FIG. 3K,
each molecular tag serves as its own standard for identifying peak
locations. The figure shows an electropherogram having ten peaks,
mT.sub.1 through mT.sub.10. Each of the peaks comprises signal
contributions from molecular tags released in the assay and
molecular tag standards (280). As shown with molecular tags,
mT.sub.2 (282) and mT.sub.5 (284), when no molecular tag is
released in the assay, then the observed peak is entirely due to
the standard, which is present in a known and detectable
quantity.
Peak Identification and Correlation
[0117] After an electropherogram data set is read by a processor,
each peak in the data is identified, or located, by a single
migration time. In the process of identifying peaks, conventional
smoothing or filtering algorithms may be applied to remove noise
and outlying data points that have no physical relevance, e.g.
using moving average filters, Savitzky-Golay filters, or the like.
Algorithms for such filters are disclosed in the following
references: Numerical Recipes in C: The Art of Scientific Computing
(Cambridge University Press, Cambridge, 1992); Hamming, Digital
Filters, Second Edition (Prentice-Hall, Inc., Englewood Cliffs,
N.J., 1983); and the like. Conventional peak identification
algorithms may be employed to determine the locations and sizes of
all peaks in the electropherogram data. A preferred peak
identification algorithm is disclosed more fully below. As
illustrated in FIG. 3D, the number of peaks identified may be
larger than the number of molecular tags used in an assay. In the
example of FIG. 3D, 22 peaks are identified, while only six
molecular tags are used in the assay. Since the molecular tags and
standards are predetermined molecules, their migration times under
standardized conditions may be determined beforehand empirically.
Thus, for each molecular tag, an interval may be defined (referred
to herein as a "migration interval"), as illustrated in FIG. 3D by
the shaded rectangles below the electropherogram. The width of the
migration interval may be defined in a variety of ways. For
example, the center of each interval may correspond to an
empirically determined mean value, referred to herein as the
"empirical migration time" (shown as a vertical line in the shaded
rectangles in the Figure), and the width of the interval may be
taken as twice the standard deviation, optionally multiplied by a
user-defined value. Peaks whose locations fall outside of the
migration intervals may be disregarded, as illustrated in FIG. 3E.
In some intervals, e.g. (217) and (219), more than one peak
location may be identified. The present invention provides a method
for selecting among such peaks to make a correct correlation with a
molecular tag.
[0118] In one embodiment of the invention, after all peaks are
identified, a first electrophoretic standard is identified by
determining the first peak that satisfied a set of necessary
conditions based on known properties of the compound used as the
standard, e.g. optical properties (it may be a different color than
the molecular tags), quantity, known range of absolute migration
times for the system used for electrophoretic separation and upon
transformation, or the like. Preferably, a first electrophoretic
standard is determined based on (i) the location of a peak within
an empirically determined range, (ii) peak height exceeding a
predetermined minimum value, and (iii) peak area exceeding a
predetermined minimum value. In a preferred embodiment of the
invention, a second electrophoretic standard is employed that has a
longer migration time than any of the molecular tags employed in an
assay, so that upon separation and transformation an
electropherogram is produced similar to that illustrated in FIGS.
3A and 3B. Once the locations of both standards are determined, in
one embodiment, migration times of molecular tags are determined as
fractions of the interval defined by the two standards, as
illustrated in FIG. 3B.
[0119] When multiple peaks have locations within the same migration
interval, as illustrated in FIG. 3E, several methods may be
employed to select a peak correlated with the molecular tag
associated with the migration interval. In one embodiment, the
location of each candidate peak is first determined relative to the
first and second electrophoretic standards in a transformed
electropherogram. For example, in as illustrated in FIG. 3F, two
peaks are located at t.sub.21 and t.sub.22 within the migration
interval centered at empirically determined, T.sub.2. The following
values are determined:
S.sub.1=(t.sub.21-T.sub.s1)/(T.sub.s2-T.sub.s1)
S.sub.2=(t.sub.22-T.sub.s1)/(T.sub.s2-T.sub.s1)
[0120] The ratio, S.sub.1 or S.sub.2, that is closest to the ratio
of the empirically determined migration time, T.sub.2, and the
difference between the migration times of the standards, that is,
T.sub.2/(T.sub.s2-T.sub.s1), determines which candidate peak is
correlated to the molecular tag of the migration interval.
[0121] In another embodiment, the location of each candidate peak
is first determined relative to second electrophoretic standard and
the previously determined peak location correlated with a molecular
tag. For example, in as illustrated in FIG. 3F, two peaks are
located at t.sub.21 and t.sub.22 within the migration interval
centered at empirically determined, T.sub.2. The following values
are determined:
S'.sub.1=(t.sub.21-T.sub.1)/(T.sub.s2-T.sub.1)
S'.sub.2=(t.sub.22-T.sub.1)/(T.sub.s2-T.sub.1)
[0122] The ratio, S'.sub.1 or S'.sub.2, that is closest to the
ratio of the empirically determined migration time, T.sub.2, and
the difference between the migration times of the second standard
and T.sub.1, that is, T.sub.2/(T.sub.s2-T.sub.1), determines which
candidate peak is correlated to the molecular tag of the migration
interval. In this embodiment, as peak locations are successively
correlated to molecular tags, the most recent such identified
migration time is used to select the next migration time when
multiple peak locations are present in a migration interval. When
no, or low levels of, molecular tag is generated in an assay, a
corresponding peak may have a low signal-to-noise ratio and its
location may be difficult to identify accurately. Therefore, for a
peak location to be used as a standard, preferably such a peak has
a signal-to-noise ratio above a minimal value. In one aspect, the
minimum signal-to-noise ratio is at least 1.5, and preferably, at
least 2.0, and more preferably, 2.5.
[0123] As mentioned above, peaks may be identified in
electropherogram data and transformed electropherogram data alike
in various ways, e.g. curve fitting, or the like. A preferred
algorithm for determining peak location and other parameters, such
as, peak height, peak size or area, and peak signal-to-noise ratio,
is illustrated in FIGS. 3G to 3J and the flowchart of FIG. 4. As
shown in FIG. 3G, a peak search window (210) is established having
width (212). Window (210) scans (214) the entire data set by
starting at the earliest (leftmost) time points, then after
carrying out peak detection and analysis steps, the window (212) is
shifted to the right a predetermined amount to an overlapping set
of times for again carrying out the peak detection and analysis
steps. This process continues until all of the data has been
analyzed. The width of window (212), the amount shifted in each
cycle of peak detection and analysis, are design choices within the
ordinary skill in the art. After the position of peak search window
(212) is established, a value for the local noise level, that is,
the noise level within the search window, is determined as
illustrated in FIGS. 3H and 31. First, an average (222) is taken of
all the data values, F(X.sub.i), in the window (220), after which
all the data values in excess of the computed average are reduced
to the average value (222), shown graphically (223) in FIG. 31.
This process is repeated and a new average value (226) is obtained.
Again, data values (224) that exceed the new average (226) are
reduced to the value of the new average. The process is repeated
until there is effectively no change in the noise value, and the
final noise value is taken as the local noise value (230) of the
peak search window, as shown in FIG. 3J. Once this value is
obtained, the peak location is taken as the ordinate, or migration
time value, X.sub.max, that corresponds to the maximum data value,
F(X.sub.j), in the peak search window; the peak starting location,
t.sub.start, (236) is the ordinate corresponding to the
intersection (232) of the noise level (230) and F(X); the peak
ending location, t.sub.end, (240) is the ordinate corresponding to
the intersection (234) of the noise level (230) and F(X); peak
width is the difference between the peak ending and the peak start;
and the peak signal-to-noise ratio is the ratio of the peak height,
F(X.sub.max), to the noise value (230). Optionally, after the peak
location is determined, the noise value may be re-computed (308,
FIG. 4) with the peak search window re-centered at X.sub.max. After
a peak location is determined, refinements in the baseline value of
the local noise may be made. For example, local noise values may be
computed adjacent to peak start and peak end points to determine
the slope of a baseline of the peak. Such a value may then be used
in computing a more accurate value of peak area. After such peak
parameters are computed, certain necessary conditions (314) must be
met before peak area is determined and the next window shift
implemented. Necessary conditions include that the peak width does
not overlap other peak widths, that the peak width is wider than a
pre-set minimum, e.g. no process were implemented to remove
spurious spikes and other outlying values from the electropherogram
data. Preferably, peak area is determined by calculating the
time-normalized area, that is, the value:
PA=E[F(X.sub.i)/X.sub.i] for i=t.sub.start, t.sub.end
Assays Analyzed by Electrophoretic Separation
[0124] Several types of assays may be employed for generating
molecular tags that are analyzed in accordance with the invention,
such as those exemplified in FIGS. 5A-5C. In FIG. 5A, the Kth
analyte (1000) in a plurality of n analytes in a sample is bound by
first binding agent (1002), an antibody in this case, having
cleavage-inducing moiety (1006) attached, which in this case is a
photosensitizer. Photosensitizer (1006) has an effective proximity
(1008) within which singlet oxygen generated by it upon
photoactivation can cleave the cleavable linkages holding molecular
tags ("T.sub.k") (1010) onto second binding agent (1004). After
photoactivation (1009), molecular tags within effective proximity
(1008) are released along with molecular tags from other binding
complexes to form mixture (1012), which is introduced (1014) into a
electrophoretic separation apparatus and separated into distinct
bands (1016). Separated tags are detected using conventional
detection methodologies. For example, if the molecular tags carry
fluorescent labels, then detection occurs after illumination by
light source (1020) and collection of fluorescence by detector
(1018). Detectable product (1026) is then detected at a detection
station as described for FIG. 5A.
[0125] In FIG. 5B, a method of generating molecular tags is
illustrated that is based on a "taqman" polymerase chain reaction
(PCR). While target polynucleotide (1030) is amplified by PCR using
primers (1032) and (1034), binding compound (1036) specifically
hybridizes (1040) to one strand of the target polynucleotide during
primer extension and is degraded by the 5'.fwdarw.3' exonuclease
activity of a DNA polymerase (1038), resulting (1042) in the
release of molecular tag (1044)(shown as "D-M-N"). After several
cycles (1046), sufficient molecular tag is released to generate a
detectable signal after electrophoretic separation. In FIG. 5C, a
method of generating molecular tags is illustrated that is based on
an "Invader" reaction. Invader probe (1052) and detection probe
(1054) specifically hybridize to target polynucleotide (1050) and
form a structure that is recognized by a cleavase (1056), after
which the nuclease activity of the cleavase releases molecular tag
(1058) leaving cleaved detection probe (1060) hybridized to the
target polynucleotide. The length and sequence of detection probe
(1054) is selected so that there is a rapid replacement (1062) of
cleaved detection probe (1060) with uncleaved detection probe
(1064), which is present in excess. As above, reaction cycles
continue (1066) until sufficient molecular tag is released to
generate a detectable signal after electrophoretic separation.
[0126] Samples containing analytes may come from a wide variety of
sources including cell cultures, animal or plant tissues,
microorganisms, or the like. Samples are prepared for assays of the
invention using conventional techniques, which may depend on the
source from which a sample is taken. Guidance for sample
preparation techniques can be found in standard treatises, such as
Sambrook et al, Molecular Cloning, Second Edition (Cold Spring
Harbor Laboratory Press, New York, 1989); Innis et al, editors, PCR
Protocols (Academic Press, New York, 1990); Berger and Kimmel,
"Guide to Molecular Cloning Techniques," Vol. 152, Methods in
Enzymology (Academic Press, New York, 1987); Ohlendieck, K. (1996).
Protein Purification Protocols; Methods in Molecular Biology,
Humana Press Inc., Totowa, N.J. Vol 59: 293-304; Method Booklet 5,
"Signal Transduction" (Biosource International, Camarillo, Calif.,
2002); or the like. For mammalian tissue culture cells, or like
sources, samples containing analytes may be prepared by
conventional cell lysis techniques (e.g. 0.14 M NaCl, 1.5 mM
MgCl.sub.2, 10 mM Tris-Cl (pH 8.6), 0.5% Nonidet P-40, and protease
and/or phosphatase inhibitors as required).
[0127] In one aspect of the present invention, sets of molecular
tags are provided that may be separated into distinct bands or
peaks by electrophoresis after they are released from binding
compounds. Molecular tags within a set may be chemically diverse;
however, for convenience, sets of molecular tags are usually
chemically related. For example, they may all be peptides, or they
may consist of different combinations of the same basic building
blocks or monomers, or they may be synthesized using the same basic
scaffold with different substituent groups for imparting different
separation characteristics, as described more fully below. The
number of molecular tags in a plurality may vary depending on
several factors including the mode of separation employed, the
labels used on the molecular tags for detection, the sensitivity of
the binding moieties, the efficiency with which the cleavable
linkages are cleaved, and the like. In one aspect, the number of
molecular tags in a plurality ranges from 2 to several tens, e.g.
50. In other aspects, the size of the plurality may be in the range
of from 2 to 40, 2 to 20, 2 to 10, 3 to 50, 3 to 20, 3 to 10,4 to
50,4 to 10,5 to 20, or 5 to 10.
Binding Compounds and Molecular Tags
[0128] An aspect of the invention includes providing mixtures of
pluralities of different binding compounds, wherein each different
binding compound has one or more molecular tags attached through
cleavable linkages. The nature of the binding compound, cleavable
linkage and molecular tag may vary widely. A binding compound may
comprise a binding moiety, such as an antibody binding composition,
an antibody, a peptide, a peptide or non-peptide ligand for a cell
surface receptor, a protein, an oligonucleotide, an oligonucleotide
analog, such as a peptide nucleic acid, a lectin, or any other
molecular entity that is capable of specific binding or complex
formation with an analyte of interest. In one aspect, a binding
compound, which can be represented by the formula below, comprises
one or more molecular tags attached to an analyte-specific binding
moiety.
B-(L-E).sub.k
[0129] wherein B is a binding moiety; L is a cleavable linkage; and
E is a molecular tag. Preferably, in homogeneous assays for
non-polynucleotide analytes, cleavable linkage, L, is an
oxidation-labile linkage, and more preferably, it is a linkage that
may be cleaved by singlet oxygen. The moiety "-(L-E).sub.k"
indicates that a single binding compound may have multiple
molecular tags attached via cleavable linkages. In one aspect, k is
an integer greater than or equal to one, but in other embodiments,
k may be greater than several hundred, e.g. 100 to 500, or k is
greater than several hundred to as many as several thousand, e.g.
500 to 5000. Within a composition of the invention, usually each of
the plurality of different types of binding compound has a
different molecular tag, E. Cleavable linkages, e.g.
oxidation-labile linkages, and molecular tags, E, are attached to B
by way of conventional chemistries.
[0130] Once each of the binding compounds is separately conjugated
with a different molecular tag, it is pooled with other binding
compounds to form a plurality of binding compounds, or a binding
composition. Usually, each different kind of binding compound is
present in such a composition in the same proportion; however,
proportions may be varied as a design choice so that one or a
subset of particular binding compounds are present in greater or
lower proportion depending on the desirability or requirements for
a particular embodiment or assay. Factors that may affect such
design choices include, but are not limited to, antibody affinity
and avidity for a particular target, relative prevalence of a
target, fluorescent characteristics of a detection moiety of a
molecular tag, and the like.
[0131] In one aspect, B is an oligonucleotide defined by the
following formula:
E-N-T
[0132] where E is as defined above, N is a nucleotide, and T is an
oligonucleotide specific for a polynucleotide analyte. Preferably,
N is attached to the 5' nucleotide of T by way of a natural
phosphodiester bond. E may be attached to N via several different
attachment sites, either on the base of N or its ribose or
deoxyribose moiety. Preferably, E is attached to the 5' carbon of N
by way of a phosphodiester bond. Synthesis of such compounds is
taught in U.S. Pat. Nos. 6,322,980 and 6,514,700, which are
incorporated by reference; and in International patent publication
WO 01/83502. In this class of binding compound, the cleavable
linkage is preferably the phosphodiester bond between N and T, and
it is cleaved by way of an enzymatic reaction by a nuclease that
recognizes specific structures formed by the binding compound, the
target polynucleotide, and possibly other molecular elements. As a
result of the enzymatic reaction molecular tag of the form "E-N"
are released. Preferably, the enzymatic reaction is in conjunction
with an amplification reaction so that in a single assay each
target polynucleotide gives rise to many hundreds, or thousands, of
released molecular tags. In one aspect, molecular tags may be
generated by any one of several nucleic acid-based signal
amplification techniques that use the degradation of a probe with a
nuclease activity, including but not limited to "taqman" assays,
e.g. Gelfand, U.S. Pat. No. 5,210,015; probe-cycling assays, e.g.
Brow et al, U.S. Pat. No. 5,846,717; Walder et al, U.S. Pat. No.
5,403,711; Hogan et al, U.S. Pat. No. 5,451,503; Western et al,
U.S. Pat. No. 6,121,001; Fritch et al, U.S. Pat. No. 4,725,537;
Vary et al, U.S. Pat. No. 4,767,699; and other degradation assays,
e.g. Okano and Kambara, Anal. Biochem., 228: 101-108 (1995).
Exemplary released molecular tags of this embodiment are
illustrated in FIGS. 6A and 6B. In this embodiment, released
molecular tags preferably have the form "(M, D)-N", where the
moiety "(M, D)" is defined as described below.
[0133] In another aspect, B is an antibody binding composition.
Such compositions are readily formed from a wide variety of
commercially available antibodies, both monoclonal and polyclonal,
specific for a wide variety of analytes. Extensive guidance can be
found in the literature for covalently linking molecular tags to
binding compounds, such as antibodies, e.g. Hermanson, Bioconjugate
Techniques, (Academic Press, New York, 1996), and the like. In one
aspect of the invention, one or more molecular tags are attached
directly or indirectly to common reactive groups on a binding
compound. Common reactive groups include amine, thiol, carboxylate,
hydroxyl, aldehyde, ketone, and the like, and may be coupled to
molecular tags by commercially available cross-linking agents, e.g.
Hermanson (cited above); Haugland, Handbook of Fluorescent Probes
and Research Products, Ninth Edition (Molecular Probes, Eugene,
Oreg., 2002). In one embodiment, an NHS-ester of a molecular tag is
reacted with a free amine on the binding compound.
[0134] When L is oxidation labile, L is preferably a thioether or
its selenium analog; or an olefin, which contains carbon-carbon
double bonds, wherein cleavage of a double bond to an oxo group,
releases the molecular tag, E. Illustrative thioether bonds are
disclosed in Willner et al, U.S. Pat. No. 5,622,929 which is
incorporated by reference. Illustrative olefins include vinyl
sulfides, vinyl ethers, enamines, imines substituted at the carbon
atoms with an .alpha.-methine (CH, a carbon atom having at least
one hydrogen atom), where the vinyl group may be in a ring, the
heteroatom may be in a ring, or substituted on the cyclic olefinic
carbon atom, and there will be at least one and up to four
heteroatoms bonded to the olefinic carbon atoms.
[0135] Molecular tag, E, is preferably a water-soluble organic
compound that is stable with respect to the active species,
especially singlet oxygen, and that includes a detection or
reporter group. Otherwise, E may vary widely in size and structure.
In one aspect, E has a molecular weight in the range of from about
50 to about 2500 daltons, more preferably, from about 50 to about
1500 daltons. Preferred structures of E are described more fully
below. E may comprise a detection group for generating an
electrochemical, fluorescent, or chromogenic signal. Preferably,
the detection group generates a fluorescent signal. Electrophoretic
standards of the invention may be selected from the same set of
compounds as are the molecular tag. In one aspect, one or more
molecular tags in a plurality may be designated and used as
electrophoretic standards in the method of the invention. When used
as an electrophoretic standard, a known quantity of the molecular
tag is added to the mixture to be separated. That is, molecular
tags used as electrophoretic standards are not released from a
binding compound, they are prepared in their released form and
added directly to the mixture to be separated.
[0136] Molecular tags within a plurality are selected so that each
has a unique electrophoretic separation characteristic and/or a
unique optical property with respect to the other members of the
same plurality. In one aspect, the electrophoretic separation
characteristic is migration time under set of standard separation
conditions conventional in the art, e.g. voltage, capillary type,
electrophoretic separation medium, or the like. In another aspect,
the optical property is a fluorescence property, such as emission
spectrum, fluorescence lifetime, fluorescence intensity at a given
wavelength or band of wavelengths, or the like. Preferably, the
fluorescence property is fluorescence intensity. For example, each
molecular tag of a plurality may have the same fluorescent emission
properties, but each will differ from one another by virtue of a
unique migration time. On the other hand, or two or more of the
molecular tags of a plurality may have identical migration times,
but they will have unique fluorescent properties, e.g. spectrally
resolvable emission spectra, so that all the members of the
plurality are distinguishable by the combination of molecular
separation and fluorescence measurement.
[0137] Preferably, released molecular tags are detected by
electrophoretic separation and the fluorescence of a detection
group. In such embodiments, molecular tags having substantially
identical fluorescence properties have different electrophoretic
mobilities so that distinct peaks in an electropherogram are formed
under separation conditions. Preferably, pluralities of molecular
tags of the invention are separated by conventional capillary
electrophoresis apparatus, either in the presence or absence of a
conventional sieving matrix. Exemplary capillary electrophoresis
apparatus include Applied Biosystems (Foster City, Calif.) models
310, 3100 and 3700; Beckman (Fullerton, Calif.) model P/ACE MDQ;
Amersham Biosciences (Sunnyvale, Calif.) MegaBACE 1000 or 4000;
SpectruMedix genetic analysis system; and the like. Electrophoretic
mobility is proportional to q/M.sup.2/3, where q is the charge on
the molecule and M is the mass of the molecule. Desirably, the
difference in mobility under the conditions of the determination
between the closest electrophoretic labels will be at least about
0.001, usually 0.002, more usually at least about 0.01, and may be
0.02 or more. Preferably, in such conventional apparatus, the
electrophoretic mobilities of molecular tags of a plurality differ
by at least one percent, and more preferably, by at least a
percentage in the range of from 1 to 10 percent.
[0138] In one aspect, molecular tag, E, is (M, D), where M is a
mobility-modifying moiety and D is a detection moiety. The notation
"(M, D)" is used to indicate that the ordering of the M and D
moieties may be such that either moiety can be adjacent to the
cleavable linkage, L. That is, "B-L-(M, D)" designates binding
compound of either of two forms: "B-L-M-D" or "B-L-D-M."
[0139] Detection moiety, D, may be a fluorescent label or dye, a
chromogenic label or dye, an electrochemical label, or the like.
Preferably, D is a fluorescent dye. Exemplary fluorescent dyes for
use with the invention include water-soluble rhodamine dyes,
fluoresceins, 4,7-dichlorofluoresceins, benzoxanthene dyes, and
energy transfer dyes, disclosed in the following references:
Handbook of Molecular Probes and Research Reagents, 8th ed.,
(Molecular Probes, Eugene, 2002); Lee et al, U.S. Pat. No.
6,191,278; Lee et al, U.S. Pat. No. 6,372,907; Menchen et al, U.S.
Pat. No. 6,096,723; and Lee et al, U.S. Pat. No. 5,945,526. More
preferably, D is a fluorescein or a fluorescein derivative.
[0140] Other aspects and advantages of the present invention will
be understood upon consideration of the following illustrative
examples.
EXAMPLE 1
[0141] This example illustrates the use of integrated current to
transform an electropherogram to reduce the variation in the
identification of electropherogram peak positions. Ninety samples
containing a multiplex of ten molecular tags were analyzed by
capillary electrophoresis, wherein the molecular tags were each
present in varying amounts. The peaks of the tags in the resulting
electropherograms were analyzed according to the methods of the
present invention and compared with a standard analysis method
employing added standards to calibrate the migration such as
described by Williams et al. in U.S. Patent Application No.
2003/0170734 A1. For visual clarity in the figures only a section
of the electropherograms are presented, however similar conclusions
were obtained for all of the analytes as shown below.
[0142] Sample solutions containing the ten molecular tags shown in
FIGS. 6A and 6B were prepared in 10 .mu.L volumes, also containing
10 mM N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid
(TAPS), 6.25 mM MgCl.sub.2, 0.25% Tween 20 and 0.25% NP-40. The 10
.mu.L samples were transferred to an injection plate and analyzed
by capillary electrophoresis using a MegaBACE 1000 (Amersham
Biosciences, Piscataway, N.J.). Capillary columns as provided by
the manufacturer were charged with POP4 separation matrix (Applied
Biosystems, Foster City, Calif.) and a running buffer of 100 mM
TAPS. The operating conditions were: injection using 15 kV for 80
s, separation using 15 kV for 60 min, with the temperature held at
30.degree. C. Analytes were detected by laser-induced fluorescence
(LIF) using an Ar.sup.+ ion laser (488 nm) for excitation, with the
detector input filtered with a 520 nm (+/-5 nm) band pass filter.
The current was recorded with the current measuring unit of the
instrument. The fluorescent signal and the current in each
capillary were sampled at 1.67 Hz.
[0143] The electrophoretic separation was performed under constant
voltage, and the current was recorded as a function of time for
each capillary. FIG. 7A shows an expanded section of the
electropherograms for seven of the ninety samples represented as
the signal versus time. The expanded section features seven peaks
associated with seven of the molecular tags. As can be appreciated
from the graph, the observed peak position for any of the analytes
varies considerably among the electropherograms.
[0144] A common method for further improving reproducibility is the
inclusion of electrophoretic migration standards, by which the
relative migration distance or migration time of the analytes is
determined. In this example, two electrophoretic standards that
were added to the sample were used to determine the relative peak
locations of the analytes. One standard migrated faster than the
analytes and was assigned a mobility of 0 (zero), while the other
standard migrated slower than the analytes and was assigned a
mobility of 1 (one). The peak position of each analyte was
interpolated between the standards to determine its relative peak
location, expressed as a decimal number between 0.0 and 1.0. FIG.
7B shows an expanded section of the same electropherograms of FIG.
7A, now plotted as the signal versus relative migration time.
[0145] In a third method, the electropherogram data sets were
analyzed according to one embodiment of the present invention
whereby the electropherograms were first transformed to the signal
as a function of the cumulative current.
[0146] More specifically, to perform the transformation the current
recorded during each of the 90 runs was first integrated to provide
the cumulative current as a function of time for each run. The
integration was performed by summing, for each time point, the
recorded, discrete data points of the current as sampled from the
beginning of the separation up to that point for each time step: 4
I c ( t ) = I 0 + i = 1 t I i ; for each t , from t = 1 to t =
T
[0147] where I.sub.c is the cumulative current, t is each sampled
time point, I.sub.0 is the current at t=0, I.sub.i is the current
sampled at the i.sup.th time point, and T is the total separation
time, to generate a data set of the series of I.sub.c as a function
of time. Then the signal was graphed as a function of the
cumulative current. The electrophoretic standards were identified,
assigned respectively the mobility values of 0 and 1, and the
relative peak locations of the analytes were determined as usual.
FIG. 7C shows the transformed electropherograms as signal versus
relative mobility for the same subset of seven runs.
[0148] The average and the variation of the observed migration
times (MT) of the ten analytes for all of the 90 electropherograms
were determined for the three analysis methods as represented in
FIGS. 7A-7C and are shown in Table 1. The first method used the
uncorrected electropherogram data of signal versus time. The second
method used electrophoretic standards to calculate relative
migration times. In the third method, the relative migration time
was determined for the transformed electropherogram data.
1 TABLE 1 Method Uncorrected Relative Transformed MT [s] MT Rel. MT
Analyte Avg. (% CV) Avg. (% CV) Avg. (% CV) A319 990.0 2.90 0.122
2.38 0.122 1.09 A317 1030.9 2.92 0.151 2.38 0.150 1.41 A95 1096.7
2.91 0.197 2.50 0.196 1.05 A410 1116.8 2.85 0.214 2.37 0.212 0.90
A281 1166.9 2.83 0.250 2.63 0.248 0.88 A388 1215.8 2.87 0.287 2.32
0.283 0.73 A405 1253.7 2.86 0.313 2.27 0.309 0.72 A324 1276.0 2.87
0.329 2.31 0.325 0.75 A322 1314.4 2.86 0.357 2.16 0.352 0.66 A386
1451.4 2.93 0.457 1.98 0.449 0.53
[0149] As demonstrated by the experiment, among the three methods,
analysis of the data using the method of transforming the
electropherogram provided the smallest coefficient of variation and
thus the most consistent determination of the peak migration times
(as the relative migration time) across the multiple samples. In
particular, the analysis provided by using standards to determine
relative migration times, a standard technique in the art, was less
reliable, showing a larger variation as evidenced by the higher %
CV. The present invention as embodied by transforming the
electropherogram based on the cumulative current increased the
fidelity of the measurement, and thus the efficiency, accuracy and
throughput of such analyses. The improvement of being able to
determine peak locations with consistency in electropherograms
obtained in different channels or runs is expected to improve the
success rate of correlating and thus identifying peaks obtained in
electrophoretic separations.
EXAMPLE 2
[0150] This example illustrates the improved capability provided by
the present invention of identifying multiple molecular tag
analytes in samples analyzed by electrophoresis. Molecular tag
analytes were generated in experiments analyzing RNA expression
levels in rats using a Rat CYP multiplexed marker panel. Samples of
rat liver total RNA isolates were analyzed in four 96-well
microtiter plates using the Rat CYP eTag.TM. (herein referred to as
molecular tags) 10-plex assay, which is a multiplexed Invader assay
reaction that releases eTag reporter molecules whenever a specified
target mRNA is present, e.g. as discussed in Williams et al. U.S.
Patent Application No. 2003/0170734 A1.
[0151] The multiplexed eTag Invader assay was carried out in
accordance with the manufacturer's instructions using a kit
obtained from the manufacturer. Briefly, 3 .mu.L of Reaction Mix
was dispensed to the wells of a 384-well assay plate, and then 2
.mu.L of Enzyme Mix was added. Samples of 5 .mu.L of total rat
liver RNA (150 ng/well) were transferred to the wells of the assay
plate. The RNA sample was the pooled total liver RNA of 1000
Sprague-Dawley rats, 8-12 weeks old (Clontech, Palo Alto, Calif.).
The plate was sealed with polypropylene self-adhesive film (VWR)
and incubated at 60.degree. C. for 16 h.
[0152] Following incubation, the plate seal was removed and 10
.mu.L of CE Separation Solution was added to the assay solutions.
The solutions were mixed and 10 .mu.L aliquots were transferred to
an injection plate and analyzed by capillary electrophoresis using
a MegaBACE 1000 (Amersham Biosciences, Piscataway, N.J.). Capillary
array columns as provided by the manufacturer were charged with
POP4 separation matrix (Applied Biosystems, Foster City, Calif.)
and a running buffer of 100 mM
N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid (TAPS).
The operating conditions were: injection using 15 kV for 80 s,
separation using 15 kV for 60 min, with the temperature held at
30.degree. C. Analytes were detected by laser-induced fluorescence
(LIF) using an Ar.sup.+ ion laser (488 nm) for excitation, with the
detector input filtered with a 520 nm (+/-5 nm) band pass filter.
The current was recorded with the current measuring unit of the
instrument. The fluorescent signal and the current in each
capillary were sampled at 1.67 Hz. Thus the electrophoresis was
performed at constant voltage, and the time-varying current was
recorded in each capillary separation path. The released molecular
tag analytes were the set of ten molecular tags illustrated in
FIGS. 6A and 6B.
[0153] The electropherogram data were analyzed by two methods. In
Method 1 the electropherograms were used as collected, i.e. signal
intensity as a function of separation time, and in Method 2 the
electropherograms were first transformed, as described by the
present invention and as described in Example 1, to
electropherograms representing the signal intensity as a function
of the cumulative current. A representative transformed
electropherogram is illustrated in FIG. 8. Then, the
electropherograms of each method were analyzed using eTag Informer
2.0 to identify the peaks corresponding to the standards and the
molecular tags. Separate databases were created for peak
identification by eTag Informer for each analysis method. Thus, the
electropherograms of each method were analyzed on the basis of
correlation to relative peak locations (with respect to two
electrophoretic standards) that were determined using the same
conditions and data types.
[0154] Table 2 reports the database entries used by eTag Informer
for each method for the relative mobility of the 10 molecular tags
comprising the Rat CYP kit. The relative peak locations were
determined by the manual analysis and averaging of eleven replicate
CE runs using the run conditions described above. Also reported are
the percentage coefficients of variation for each peak. Although
the observed variation is smaller with Method 2, and thus the
expected peak location range used by the software is somewhat
smaller, a better success rate for identifying the peaks using
Method 2 has been demonstrated, as described below.
2 TABLE 2 Relative Peak Location (% CV) eTag ID Method 1 Method 2
A319 0.115 (1.28) 0.121 (1.04) A317 0.143 (1.31) 0.151 (1.00) A95
0.186 (1.17) 0.196 (0.96) A410 0.202 (1.13) 0.212 (0.97) A281 0.232
(1.02) 0.244 (0.88) A388 0.269 (0.98) 0.283 (0.88) A405 0.294
(0.93) 0.308 (0.76) A324 0.309 (0.91) 0.325 (0.81) A322 0.335
(0.86) 0.351 (0.72) A386 0.365 (0.93) 0.381 (0.80)
[0155] The results for the four plates run for each of the two
methods of analysis are summarized in Table 3 as the total number
of peaks observed, the number of those peaks that were accurately
identified (called), and the percentage of peaks accurately
identified. In some cases the total number of peaks observed is
less than the total expected (960=10-plex.times.96 wells) due to
bubbles, injection failures and other mechanical failures. All
other peaks were otherwise included in the analysis. As illustrated
by the table, in two cases (plates C and D) the data generated by
the instrument was distorted to an extant that more than half of
the peaks could not be called by Method 1, even despite the fact
that the analysis incorporates two electrophoresis standards which
flank the set of molecular tag analytes. By contrast, using Method
2 to correct for the local conditions experienced by the sample
during the separation, the same samples (plates C and D) were
called with a greater than 95% success rate.
[0156] Furthermore, Method 2 was demonstrated to not adversely
affect the electropherogram data in runs having more normal
characteristics. For example, in the runs of plates A and B, local
effects such as sample conductivity, temperature, injection plug
discontinuity and the like did not significantly perturb the
dynamics of the separation process and thus Method 1 provided a
useful data analysis. Method 2 still provided a modest improvement
in the ability to identify the peaks in plate A, while plate B
illustrates the fact that in some instances the older methods are
adequate for analysis.
3 TABLE 3 Multiwell Sample Plate ID Plate A Plate B Plate C Plate D
Meth. 1 Meth. 2 Meth. 1 Meth. 2 Meth. 1 Meth. 2 Meth. 1 Meth. 2 No.
Peaks 741 741 741 741 910 914 910 914 No. Peaks Called 658 686 725
722 277 873 418 895 % Peaks Called 88.8 92.6 97.8 97.4 30.4 95.5
45.9 97.9
[0157] This example illustrates that the methods of the present
invention may be applied uniformly to electropherogram data sets to
improve the fidelity of the information content, such as peak
location, of the electropherogram, thus leading to better accuracy,
consistency and throughput in the analysis of electrophoretic
separations.
[0158] All publications and patent applications mentioned in this
specification are indicative of the level of skill of those skilled
in the art to which this invention pertains. All publications and
patent applications set forth herein are incorporated by reference
to the same extent as if each individual publication or patent
application was specifically and individually indicated to be
incorporated by reference.
[0159] The invention now having been fully described, it will be
apparent to one of ordinary skill in the art that many changes and
modifications can be made thereto without departing from the spirit
or scope of the appended claims.
* * * * *